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Balancing protection of plant varieties and other public interests.

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Wu, C. Balancing Protection of Plant Varieties and Other Public Interests. Sustainability 2024 , 16 , 5445. https://doi.org/10.3390/su16135445

Wu C. Balancing Protection of Plant Varieties and Other Public Interests. Sustainability . 2024; 16(13):5445. https://doi.org/10.3390/su16135445

Wu, Chenwen. 2024. "Balancing Protection of Plant Varieties and Other Public Interests" Sustainability 16, no. 13: 5445. https://doi.org/10.3390/su16135445

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• Published: 17 July 2021

Transgenic and genome-edited fruits: background, constraints, benefits, and commercial opportunities

• Maria Lobato-Gómez   ORCID: orcid.org/0000-0002-2589-6216 1 ,
• Seanna Hewitt 2 ,
• Teresa Capell 1 ,
• Paul Christou 1 , 3 ,
• Amit Dhingra   ORCID: orcid.org/0000-0002-4464-2502 2 &
• Patricia Sarai Girón-Calva   ORCID: orcid.org/0000-0003-0166-8478 1  

Horticulture Research volume  8 , Article number:  166 ( 2021 ) Cite this article

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• Metabolic engineering
• Molecular engineering in plants

Breeding has been used successfully for many years in the fruit industry, giving rise to most of today’s commercial fruit cultivars. More recently, new molecular breeding techniques have addressed some of the constraints of conventional breeding. However, the development and commercial introduction of such novel fruits has been slow and limited with only five genetically engineered fruits currently produced as commercial varieties—virus-resistant papaya and squash were commercialized 25 years ago, whereas insect-resistant eggplant, non-browning apple, and pink-fleshed pineapple have been approved for commercialization within the last 6 years and production continues to increase every year. Advances in molecular genetics, particularly the new wave of genome editing technologies, provide opportunities to develop new fruit cultivars more rapidly. Our review, emphasizes the socioeconomic impact of current commercial fruit cultivars developed by genetic engineering and the potential impact of genome editing on the development of improved cultivars at an accelerated rate.

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The conventional breeding of fruit crops can take more than two decades due to the long juvenile period of woody species 1 . Genetic engineering allows improved varieties to be developed more quickly, and the vegetative propagation of fruit trees allows the engineered cultivars to achieve coverage of larger areas than crops that depend on sexual reproduction 2 . All genetically engineered fruit crops have been produced either by Agrobacterium -mediated transformation or direct DNA transfer. In each case, the efficiency of transformation is highly dependent on the species and even cultivar, requiring the development of bespoke optimized methods consisting of efficient gene delivery, selection, and regeneration from transformed explants 2 . Most fruit tree species are highly heterozygous, and to maintain the characteristics of the original variety the transgenic events should be derived from mature tissue (such as leaves) rather than embryogenic explants 3 .

The first genetically engineered fruit product (Flavr Savr™ tomato) was deregulated in 1992 and introduced into the market in 1994 4 . A gene that triggers pectin solubilization was downregulated in the transgenic fruits, resulting in delayed fruit softening and an extended shelf-life 5 . Several additional fruit crops with traits improved by genetic engineering have received regulatory approval for commercialization in different parts of the world, and are intended for cultivation either as human food or animal feed. These are tomato ( Solanum lycopersicum ) 6 , 7 , 8 , 9 , papaya ( Carica papaya L.) 10 , 11 , pepper ( Capsicum annuum ) 12 , plum ( Prunus domestica ) 13 , eggplant ( Solanum melongena L.) 14 , apple ( Malus domestica Borkh.) 15 , melon ( Cucumis melo L.) 16 , and pineapple ( Ananas comosus L. Merr.) 17 . Most of the transgenic fruits were developed to improve agronomic productivity by conferring pest or disease resistance, or delayed ripening. However, more recent products have addressed quality traits by eliminating fruit browning or adding new visual traits such as flesh color. Some engineered fruit crops have been withdrawn from the market because they were not commercially viable (Flavr Savr™ tomato 4 , 18 ) or were never commercialized (Melon A and B 16 , 19 ).

Advances in genetic engineering, particularly the development of genome editing technologies have provided new tools for the generation of improved fruit varieties. Many proof-of-concept examples involving fruit crops have been reported and the further development and marketing of such varieties could have a major socioeconomic impact. Here we discuss the history and current status of genetically engineered fruit crops and the promise offered by genome editing. In recent years, several countries have amended their current regulations or have developed new guidelines to regulate genome-edited plants and its products 20 . This may make it possible that genome-edited fruits, similarly to all other genome-edited crops, reach the market faster in countries with a genome editing friendly policy 20 , 21 . Here, we first discuss fruit varieties that have already been approved for commercialization, focusing on those that are on the market. We then consider fruit varieties developed more recently using genetic engineering or genome editing, and their potential socioeconomic impact.

Genetically engineered fruits approved for commercialization

Trait description and drivers.

Genetically engineered fruits have been developed with unique agronomic characteristics that are often difficult to achieve by conventional breeding, and are designed to meet the specific needs of growers and/or customers. Fruits that have been developed by genetic engineering are shown in Fig. 1 . Some varieties were approved but not ultimately commercialized, or were launched but subsequently removed from the market, and these are not considered in detail.

Year indicates the year of first approval. Currently on the market indicated as light blue boxes

Papaya resistant to papaya ringspot virus

In 1992, papaya ringspot virus (PRSV) was detected in Puna, the major papaya-producing district in Hawaii. PRSV resistance was not found in papaya germplasm or in wild Carica species suitable as candidates for interspecific hybridization. Furthermore, insecticides failed to control the aphid vectors responsible for virus transmission 22 , and many orchards were therefore abandoned due to PRSV infestation 10 . The widely cultivated ‘Sunset’ papaya was transformed with a gene derived from a Hawaiian strain to produce the transgenic papaya ‘SunUp’, which is completely resistant to PRSV in Hawaii 10 . ‘SunUp’ papaya was crossed with ‘Kapoho’, a non-engineered cultivar, to obtain the yellow-flesh ‘Rainbow’ papaya, which is also resistant to PRSV 23 .

In China, PRSV has threatened the papaya industry for 50 years 24 . Similarly to the ‘SunUp’ variety, transgenic Huanong No. 1 papaya is resistant to the four predominant PRSV strains found in South China (Hainan, Guangdong, Guangxi, and Yunnan provinces), namely Ys, Vb, Sm and Lc 24 . Additionally, Huanong No. 1 produces bigger fruits with thicker flesh than the original cultivar 24 . In 2012, some Huanong No. 1 papayas grown in Hainan exhibited PRSV-like symptoms, suggesting that resistance is beginning to break. Phylogenetic analysis revealed the presence of a new virus lineage in Hainan and Guangdong papaya plantations, which may pose a threat to Huanong No. 1 papaya cultivation 25 .

Tomato and sweet pepper resistant to cucumber mosaic virus

In 1990, tomato crops in Fujian province (China) were affected by a virulent strain of cucumber mosaic virus (CMV) causing severe necrosis 26 . CMV is a major threat to tomato and sweet pepper and thus the tomato line PK-TM8805R and the sweet pepper line PK-SP01 were developed 24 . Both fruits express a CMV protein gene, conferring resistance to CMV, but data concerning the performance of these cultivars have not been published 26 .

Squash resistant to potyviruses

Like CMV, zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus 2 (WMV 2) are potyviruses transmitted by aphids. Together, these viruses can reduce the yields of squash by up to 80% 27 . Resistance to these viruses is not found in squash germplasm, and cannot be introduced by interspecific hybridization due to hybrid incompatibility and the concomitant transfer of undesirable traits 28 . In 1995, several transgenic inbred squash lines were developed by transformation with single or multiple viral protein genes from ZYMV, WMV2, and CMV. Transgenic lines ZW-20 and CZW-3 showed complete resistance to ZYMV and WMV2, line CZW-3 showed additional resistance to CMV 28 .

Eggplant resistant to eggplant fruit and shoot borer

In Bangladesh, eggplant is the second most important fruit crop and a major source of income for small, resource-poor farmers 29 . Eggplant fruits are unmarketable when infested with eggplant fruit and shoot borer (EFSB) larvae ( Leucinodes orbonalis ) but effective prevention requires the application of more than 100 sprays of insecticide each season. In addition to the detrimental impact on the environment, this accounts for more than a quarter of production costs, and there are still losses due to the prevalence of EFSB 30 . Resistant cultivars have not been developed by conventional breeding 31 , but a transgenic variety producing Bacillus thuringiensis (Bt) toxins is resistant to EFSB has been commercialized 30 . Infestations of the Bt variety occur at a frequency of 0.04–0.88% compared to 48–57% for the equivalent non-transgenic cultivar. In 2019, the average yield of Bt eggplant in Bangladesh was 19.8 t/ha, compared to 16.6 t/ha for the non-transgenic cultivar 29 .

Non-browning apple

Fruit quality is affected by the activity of polyphenoloxidases (PPOs), which oxidize phenolic compounds and cause gradual browning in fleshy fruits such as apple. PPOs are activated by exposure to oxygen, resulting in browning when fruits are damaged, peeled, or cut. Enzymatic browning can be prevented by storage in an air-free environment, the inactivation of PPOs by irradiation, or through the use of chemical inhibitors and natural antioxidants 32 . The Arctic ® apple concept was developed by silencing of PPOs 33 , 34 . Currently, there are three commercial varieties of Arctic ® apple: Arctic ® Golden Delicious, Arctic ® Granny Smith, and Arctic ® Fuji. Commercial harvest of Arctic ® Golden Delicious and Arctic ® Granny Smith started in 2016, and Arctic ® Fuji will be on the market in 2021 35 .

Pink-fleshed pineapple

Fruits with different skin and flesh colors have been developed by conventional breeding 36 and in proof-of-concept engineering experiments 37 . In 2005, the Pinkglow™ transgenic pineapple was developed, in which the pink flesh accumulates lycopene due to the modification of the carotenoid pathway 17 . The skin of the Pinkglow™ pineapple also has a combination of green, yellow, orange, and red colors, whereas conventional pineapple is green and yellow. In addition to the modulation of carotenoid accumulation, an endogenous ethylene biosynthesis gene was suppressed to control flowering, but this trait has yet to be evaluated 17 .

Development of commercial transgenic fruits (currently on the market)

In 1986, the coat protein of a Hawaiian PRSV isolate was cloned at Cornell University in collaboration with the Asgrow Seed Company. The USDA Section 406 grant program supported the development of transgenic PRSV-resistant papaya with the aim to control PRSV in Hawaii. In 1992, the first PRSV-resistant papayas were developed through a collaboration involving Cornell University, University of Hawaii and the Asgrow company 10 . The University of Hawaii established the protocol for papaya transformation by particle bombardment using zygotic embryos as the starting material 10 , 38 , whereas Huanong No. 1 papaya was generated using an Agrobacterium -mediated procedure established by an independent laboratory 11 . Transgenic papaya resistant to PRSV were developed using a pathogen-derived resistance approach, in which the resistance is mediated via RNA post-transcriptional gene silencing. The underpinning mechanism involves the expression of a partial or full pathogen gene sequence in the host to disrupt the pathogen’s replication 39 . ‘SunUp’ and ‘Rainbow’ papaya contain the coat protein gene from the mild PRSV HA 5-1 isolate 10 . The coat protein is required for virus survival outside the cell and for aphid transmission 40 . The required RNA specificity explains why PRSV-resistant transgenic papaya shows a narrow spectrum of resistance to particular PRSV isolates 41 . Huanong No.1 contains the replicase protein domain (NIb) from the PRSV Ys isolate, the most prevalent strain in China in 1994 24 . The N1b and N1a proteins are needed for virus replication 40 .

Seminis Vegetable Seeds and Monsanto Company developed transgenic virus-resistant squashes in 1995 27 . ZW-20 and CZW-2 virus-resistant squashes were generated using an Agrobacterium -mediated transformation protocol 28 PTGS has been also used to produce ZW-20 and CZW-3 squash. Specifically, these lines contain the coat protein gene from FL isolates of ZYMV and WMV2, and line CZW-3 contains in addition the coat protein gene from CMV strain C 28 .

In 2000, the Maharashtra Hybrid Seeds Company (Mahyco) started to develop Bt eggplant with the collaboration of Monsanto, in India. In 2003, the Agricultural Biotechnology Support Project II (ABSPII) funded a partnership between Mahyco, Cornell University, the US Agency for International Development (USAID), and public-sector partners in India, Bangladesh, and the Philippines to develop and commercialize Bt eggplant. Under the ABSPII agreement, the EE-1 eggplant event, resistant to EFSB, was donated to the public Bangladesh Agricultural Research Institute (BARI) by Mahyco via a public–private partnership 30 . EFSB resistance was incorporated into nine local eggplant lines by BARI. The ASBPII project ended in 2014 and the distribution of Bt eggplant to farmers in Bangladesh was funded by the South Asia Eggplant Improvement Partnership (SAEIP), which comprises BARI, Cornell University, USAID, the University of the Pihilippines Los Banos, and Allience for Science 14 , 30 . Mahyco also set up its own eggplant transformation pipeline. Cotyledons from eggplant seedlings were used as explants for Agrobacterium -mediated transformation with the Bt cry1Ac gene, producing the EE-1 transgenic variety 42 .

Okanagan Specialty Fruits developed Arctic ® Apple events GD743 (Golden Delicious), GS784 (Granny Smith) 33 and GS784 (Fuji) 35 using their patented method to limit quinone biosynthesis 43 . Quinones are produced from diphenols in a reaction catalyzed by PPO, and their condensation with amino acids and proteins generates lignin-like compounds that cause browning. Cell damage is needed for plastidial PPO to act on vacuolar substrates, which is why browning only occurs in cut or otherwise damaged fruit 43 . RNA interference (RNAi) technology was used to target four apple PPO genes by expressing a chimeric sense RNA containing partial coding sequences of PPO2 , GPO3 , APO5 and pSR7 , leading to the generation of dsRNA and the suppression of homologous genes by post-transcriptional silencing 32 .

Del Monte started to develop the Pinkglow™ pineapple by modulating the carotenoid pathway 44 . ‘MD2’, also known as the Del Monte Gold pineapple, is a commercial variety developed by the company and was used as starting material. Ten years later, this transgenic pineapple was patented in the US 17 . Del Monte also patented the transformation method, which involved the cultivation of organogenic pineapple cells with A. tumefaciens . Conventional pineapple on the market has yellow flesh, reflecting the β-carotene content. The Pinkglow™ pineapple expresses the tangerine ( Citrus reticulata ) PSY gene, which is a rate-limiting enzyme in carotenoid biosynthesis during fruit development 17 . In addition, the endogenous lycopene β and ε cyclase genes ( βLYC and εLYC ) were suppressed by RNAi 17 . Ethylene promotes flowering in pineapple, and 1-aminocyclopropane-1 carboxylic acid (ACC) is the immediate ethylene precursor in plants 45 . A meristem-specific ACC synthase (ACS) was suppressed by RNAi in the Pinkglow™ pineapple to inhibit flowering 17 .

Regulatory approval and commercialization of improved fruit crops

The USA has issued the most approvals for transgenic fruit cultivation either for human consumption or as animal feed. Like other genetically engineered crops, three government agencies are responsible for the oversight of transgenic fruit cultivation and import: the US Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS), the US Environmental Protection Agency (EPA), and the US Food and Drug Administration (FDA), which is part of the Department of Health and Human Services. Depending on its characteristics, a genetically engineered fruit may fall under the jurisdiction of one or more of these agencies 46 . APHIS regulates the environmental release of genetically engineered organisms that may pose a risk to plant health, the EPA oversees pesticides, including genetically engineered plants expressing plant incorporated protectants (PIP), and the FDA ensures the safety of all human food and animal feed (also from plant origin).

In 2020, APHIS published a revision of its 1987 biotechnology regulations 47 . The new framework, known as the SECURE rule (Sustainable, Ecological, Consistent, Uniform, Responsible, and Efficient) differs from the previous regulatory framework by focusing on an organism’s properties and not on the production method 47 .

Flavr Savr™ tomato developed by Monsanto Company was the first genetically engineered fruit to gain non-regulated status from APHIS and approval by the FDA 5 , 18 . Flavr Savr™ was also approved for import into Mexico in 1995 by the Federal Commission for the Protection against Sanitary Risk (COFEPRIS), a decentralized organ of the Mexican Secretariat of Health that oversees the safe release and import of genetically engineered plants 48 . COFREPIS also permitted the import of the engineered tomato varieties Da, B, F, and Endless summer. Similarly, in 1995 Health Canada and Agriculture and Agri-food Canada determined that the Flavr Savr™ tomato was safe for human consumption and did not pose risks as a plant pest 49 . In Canada, the Flavr Savr™ tomato was marketed under the brand name MacGregor, allowing consumers to make an informed choice 49 . Flavr Savr™ was removed from the market in 1997 because the fruits were less firm than expected and the costs of production were uncompetitive 18 .

APHIS deregulated additional engineered tomato lines in the 1990s, namely Da, B, F developed by Zeneca and Petoseed Company; 35-1-N developed by Agritope, Inc; and 5345 and 8338 “Endless summer” developed by the Monsanto Company 6 , 7 , 8 , 9 , 50 . These lines were also approved as food and feed. The Da, B, and F lines were intended for processing 4 . Between 1996 and 1999, more than 1.8 million cans derived from hybrids of the F line were sold in the UK 18 , but from 1998 onwards were no longer used as food ingredients 18 . In 2000 Health Canada also approved line 5345, which was resistant to insect pests, but it has not been released onto the market 51 .

In 1999, Agritope was granted FDA approval of the Melon A and B lines for use as food 16 . The company also requested the deregulation of these lines, but withdrew the APHIS petition the same year 19 , and neither line has been commercialized.

The Pinkglow™ pineapple received FDA approval in 2016 and was marketed for the first time in October 2020 by Fresh del Monte 52 , 53 . This cultivar is grown on a single farm in Costa Rica. The C5 plum (HoneySweet) developed by the US Department of Agriculture, which is resistant to plum pox virus (PPV), has also been deregulated by APHIS, approved by the FDA and registered by the EPA 54 . It was patented in the US in 2004, but no trees have been planted thus far and it is therefore not on the market. On request, the Agricultural Research Service (the research branch of the USDA) can freely provide a limited number of heat-treated bud wood samples to be used as a genetic resource for the breeding of PPV-resistant varieties 55 .

Genetically engineered squash has been on the US market for 25 years. CZW3 squash is also approved for import as food by Health Canada 56 . The cultivation of genetically engineered papaya in the US began in 1996, and the current predominant variety is ‘Rainbow’ because it has yellow fruit flesh favored by consumers 4 . Canada and Japan are the major importers of genetically engineered papaya produced in the US, although it is also approved for cultivation in Japan 57 . Two additional papaya lines resistant to PRSV were approved for cultivation by APHIS: 63-1 developed by Cornell University and the University of Hawaii 58 , and X17-2 developed by the University of Florida, respectively 59 . Neither lines have been commercialized 4 .

Arctic ® apples were developed by Okanagan Specialty Fruits Company in Canada, and the Golden Delicious, Granny Smith, and Fuji varieties have received approval for cultivation, human consumption and use as animal feed in both Canada and the US 15 , 60 , 61 , 62 . However, Arctic ® apples are only grown in the US, and it is unclear if Artic varieties are among the 206,259 tons of apples (including dried apples) imported to Canada, most of which are grown in the US 63 , 64 .

In China, the commercialization of all genetically engineered crops is regulated by the Ministry of Agriculture (MOA) 65 , with safety advice provided mainly by the Biosafety Management Division of the Center for Science and Technology Development (CSTD) and the National Biosafety Committee (NBC). The NBC can recommend safety certification based on product testing and field trials, but only the MOA can formally provide regulatory clearance 25 . After registration, genetically engineered crops can be cultivated and commercialized but approval for commercialization is only granted at the province/region level and not nationwide.

Huafan No. 1 tomato developed by Huazhong Agricultural University was the first genetically engineered fruit to be approved for cultivation, human consumption and use as animal feed in China, followed by Da Dong No. 9 (Institute of Microbiology, CAS) and PK-TM8805R (Beijing University) tomatos 26 . Huafan No. 1 and Da Dong No. 9 are no longer cultivated in China, and the status of PK-TM8805R is unclear 26 . Similarly, the genetically engineered sweet pepper PK-SP01 developed by Beijing University was approved for cultivation and for human consumption, but the extent of its cultivation is unclear 26 . PRSV-resistant papaya Huanong No. 1 was approved for cultivation in 2006 and is commercially available in China.

In Bangladesh, the National Committee on Biosafety (NCB) grants regulatory approvals for all genetically engineered crops, assisted by a Biosafety Core Committee (BCC) 66 . The eggplant varieties Bari Bt Begun 1, 2, 3, and 4 were approved for cultivation and food use in Bangladesh, and in 2020 they are the only genetically engineered fruit commercialized in this country 29 , 30 .

Socioeconomic impact of commercialized fruits with improved traits

The socioeconomic impact of genetically engineered fruits is growing with the scale of cultivation, although less than 0.01% of the 185.43 million ha cultivated with genetically engineered crops in 2018 was represented by fruits 67 . Production and adoption rate details are provided in Table 1 . PRSV-resistant papaya is the most widely cultivated genetically engineered fruit, followed by Bt eggplant, virus-resistant squash, Arctic ® apples, and Pinkglow™ pineapple.

Virus-resistant fruits

China grew 9600 ha of PRSV-resistant papaya in 2018. Initial plantings took place in the southern Guangdong Province in 2006, but Hainan Island became the leading location for PRSV-resistant papaya production in 2017 (46%), followed by Guangdong (36%) and Guangxi (18%) provinces 57 . CMV-resistant sweet pepper and tomato have been cultivated in China since 1998 and 1999, respectively, in Beijing municipality and in Fujian and Yunnan provinces, but the scale of cultivation is unclear 26 . Data on the profitability of PRSV-resistant papaya have not been published by the Chinese authorities, so the socioeconomic impact is difficult to judge 68 .

In the US, PRSV-resistant papaya has been commercially grown in Hawaii since 1999 and it has prevented the collapse of the Hawaiian papaya industry due to the prevalence of PRSV in orchards of conventional varieties 23 . In 1992, when PRSV was first detected on Hawaii, the Puna district produced 95% of all Hawaiian papaya grown (~24,000 tons) but yields had fallen to ~12,000 tons in 1998. Two years after the introduction of the resistant variety, yields recovered to ~18,000 tons 23 . Although lower than 1992 levels, the lack of production was not caused by the virus but by the falling demand from Japan, resulting in the papaya cultivation area in Hawaii declining from more than 500 ha in 2015 to only 250 ha in 2018 4 , 67 . The shrinking Japanese market partly reflected the reluctance of retailers to handle genetically engineered products and partly the increased competition from Philippine papaya growers 4 . Nevertheless, the yield of genetically engineered papaya in 2018 was 17% higher than conventional papaya, with a net farm income gain of $2623/ha. Overall, the accumulated farm income benefit between 1999 and 2018 was $38.4 million 67 . Cultivation of PRSV-resistant papaya in Hawaii has also reduced the threat of PRSV in the Puna district, allowing papaya growers to cultivate non-transgenic varieties alongside the genetically engineered crop 23 .

Virus-resistant squash has been commercially grown in the US since 2004, mainly in Florida and Georgia. In 2018, virus-resistant squash was planted on 1000 ha, representing 6% of total squash production in the US 67 . The genetically engineered varieties achieve higher yields than conventional squash, resulting in a net gain to farmers of $10.1 million. Overall, the cumulative farm income benefit between 2004 and 2018 was $310.9 million 67 .

Insect-resistant fruit crops

Bt eggplant was first grown commercially in Bangladesh in 2014, and was cultivated on 2975 ha in 2018 67 . Eggplant is mostly grown by resource-poor farmers, who can obtain seed at no or minimal cost from three organizations: BARI, the Department of Agricultural Extension, and the Bangladesh Agricultural Corporation. Accordingly, the cost of this technology to the farmers is near zero 29 . The Bt eggplant was initially provided to 20 farmers, but by 2018, the variety had been adopted by 20,695 farmers 29 . Bt eggplant achieved 20% higher yields than conventional eggplant in 2018, and the enhanced quality resulted in a 10% increase in price. As a result, farm income has increased by $616–704/ha 29 , 67 .

As well as the direct income gains, Bt eggplant also helps to reduce pesticides. In 2016, farmers in 35 districts cultivating Bt eggplant spent 61% less on pesticides compared to farmers growing conventional varieties 69 . This difference solely represents the cost of pesticides to control EFSB because different chemicals are used to control other pests. However, the prevention of damage caused by EFSB also reduces infestations by secondary pests such as leaf-eating beetles, thrips, whitefly, mites, leaf wing bugs, and leaf roller, by 42–60% 70 .

Fruits with enhanced quality traits

Arctic ® apples were first planted in 2016 (70,000 trees planted over 80 ha). This had grown to 300,000 trees over 101 ha by 2018 and in 2019 the cultivated area exceeded 500 ha 71 . Although the profitability of growing this variety has not been made public, Okanagan Specialty Fruits states that Arctic ® apples are more suitable for mechanical harvesting and suffer less impact from finger bruising, bin rubs and other superficial damage, which results in higher packouts (an industry measure of fruit suitable for market) and therefore less waste, and similar benefits for retailers 72 . Furthermore, the Arctic ® Golden variety does not require warm packing, reducing the cost of production. Del Monte commercialized the Pinkglow™ pineapple in October 2020 so the socioeconomic impact of this variety will not be known until market data are available.

Technological advances in gene functional analysis and genetic modification of fruits

Genetic engineering can be used to investigate the functions of genes and to exploit these functions for the improvement of traits such as biotic and abiotic stress tolerance, flowering time, ripening, fruit flavor, and nutrient content. In this section, we discuss genetic engineering and genome editing technologies that have been used for the enhancement of target traits in fruit crops, which may facilitate commercialization in the future (Table 2 ). Use of CRISPR and associated genome editing technologies for the development or enhancement of fruit crops may open the door to new commercial opportunities, potentially circumventing restrictions on GM crops in many parts of the world 20 . While marketability will vary by country, additional, transgene-free cultivars may be accessible to consumers in the near future 20 , 73 , 74 .

Pathogen and pest resistance

Pathogens and pests are severe constraints affecting the growth and development of fruit trees, the development and ripening of fruits, and the quality of fruit products. In 2017 up to 30% of the fruit and vegetables losses worldwide were pre-harvest, mainly caused by pests and pathogens 75 . In many cases, conventional breeding for resistance is not possible because strong resistance is not present in available germplasm and the introgression process would take too long 2 . One strategy to enhance disease resistance in fruit crops is the modification of receptors that directly interact with or perceive the presence of a specific pathogen. In apple, overexpression of the HcrVf2 gene encoding such a receptor resulted in near-complete resistance to fungal scab ( Venturia inaequalis ) 76 . Recently, CRISPR/Cas9-mediated inactivation of the susceptibility-associated gene DspA/E-interacting protein ( DIPM4 ), also encoding a receptor, significantly reduced bacterial fire blight ( Erwinia amylovora ) symptoms by 50% in apple 77 .

Another strategy for the mitigation of pathogen symptoms is the targeting of response pathways (innate immunity) in the host. For example, the nonexpressor of pathogenesis-related 1 ( NPR1 ) gene encodes a transcriptional regulator of pathogenesis-related (PR) protein genes as part of the salicylic acid-dependent systemically acquired resistance (SAR) pathway. Sweet orange trees ( Citrus sinensus ) overexpressing NPR1 under the control of the phloem-specific SUC2 promoter exhibited enhanced resistance to huánglóngbìng (citrus greening disease), and up to 46% of the engineered plants remained disease-free for 2 years 78 . These findings highlight the importance of promoter selection in overexpression studies and indicate that NPR1 possesses a conserved role among tree fruit species in the response to pathogens.

Other PR-associated proteins have been targeted for modification in banana, chili pepper, and citrus in order to mitigate the effect of bacterial and fungal pathogens. In banana, the induction of a hypersensitive response (HR) by the overexpression of genes encoding an HR-assisting protein and a plant ferredoxin-like protein conferred resistance to banana Xanthomonas wilt, with 50–60% of the transgenic plants displaying no disease symptoms following inoculation 79 . Overexpression of the pepper carboxylesterase gene in chili pepper reduced infections by anthracnose fungus from 70% in wild-type plants to 20% 80 . Similarly, expressing the J1-1 gene encoding an antifungal defensin reduced the frequency of anthracnose lesions by up to 90% 80 , 81 . CRISPR/Cas9 was used to inactivate the grapefruit lateral organ boundary domain family protein 1 and orange WRKY22 genes, which regulate immunity responses, improving resistance to canker caused by Xanthomonas citri subsp. citri ( Xcc ) in Duncan grapefruit ( Citrus ✕ paradisi ) and Wanjincheng orange ( Citrus sinensis (L.) Osbeck) 82 , 83 , 84 , 85 . The CRISPR-induced mutation rate in grapefruit was 23–89%, and Xcc resistance was correlated with the mutation rate, as shown by the corresponding range of canker symptoms 85 . Similar findings were reported for orange plants with mutations in the WRKY22 gene 83 .

In addition to the knockout of host genes to improve pathogen and pest resistance, pathogen-derived transgenes (or other heterologous genes) serve as additional routes for the improvement of fruit traits. In pear, the expression of a bovine lactoferrin gene, which encodes a bactericidal glycoprotein, reduced fire blight symptoms by 78% compared to controls 86 . In sweet orange, expression of the E. amylovora hairpin protein triggered HR in the host plants and reduced susceptibility to citrus canker by up to 79% 87 . The expression of a synthetic insect antimicrobial peptide (cecropin B) in blood orange improved long-term resistance to huánglóngbìng by 85–100% 88 .

An important strategy in the fight against viral diseases is the expression of non-translatable pathogen genes to elicit a PR response or to silence viral components essential for replication, packaging, or systemic spreading. RNAi-mediated silencing of viral components has been achieved in banana, resulting in the complete absence of bunchy top virus disease symptoms in transgenic plants 6 months after challenge 89 . Similarly, transgenic melon and watermelon ( Citrullus lanatus ) lines displayed up to 100% resistance when challenged with several cucurbit viruses 90 , 91 , and grafted transgenic plum lines remained resistant to PPV for more than 9 years 92 . In cucumber, the CRISPR/Cas9 system was used to mutate the eukaryotic translation initiation factor 4E gene, which is associated with CMV susceptibility, resulting in 100% virus-free fruits in the T3 generation 93 . Bt cry genes have been expressed in kiwifruit ( Actinidia chinensis ) and walnut ( Juglans regia ) to protect them against insect pests, resulting in 75–100% insect pest mortality 94 .

Abiotic stress tolerance

Abiotic factors, such as drought, are also among the main factors causing pre-harvest losses of fruit and vegetables 75 . The engineering of abiotic stress tolerance in fruit trees allows them to be grown in environments where temperatures are sub-optimal, water is scarce, or high concentrations of salt and/or heavy metals in the soil are toxic and prevent the uptake of water and nutrients. Overexpression of the Na + /H + cation antiporter gene NHX1 in apple and kiwifruit prolonged survival in saline conditions by allowing the accumulation of higher concentrations of antioxidant flavonoids (60% more than normal) as well as sodium and potassium (2x more than normal) thus delaying the stress response 95 , 96 . In chili pepper, the expression of a tobacco osmotin gene increased yields by 31% accompanied by higher levels of proline, chlorophyll and reactive oxygen species (ROS) scavengers, as well as a higher relative water content 97 . Transgenic citrumelo ( Citrus paradise × Poncirus trifoliata ) plants overexpressing the enzyme Δ1-pyrroline-5-carboxylate synthase, required for proline synthesis, showed a 2.5-fold increase in drought tolerance, as determined by turgor pressure maintenance, stomatal conductance, photosynthetic rate, and transpiration rate 98 .

Fruit crops are often threatened by cold temperatures, which affect plant growth as well as the quality of maturing and ripening fruits. Cold tolerance is therefore an important target in commercial fruit development programs. In apple, overexpression of the transcription factor MYB4, which regulates cold-induced dormancy and stress pathways, allowed the transgenic plants to tolerate cold temperatures for long periods while maintaining normal water content, reflecting the accumulation of glucose, fructose, and sucrose to levels 30–38% higher than normal 99 . Overexpression of the Arabidopsis dehydration response element-binding 1b protein in grapevine reduced cold-induced wilting by 73% 100 . Similarly, the expression of a Poncirus trifoliata basic helix-loop-helix protein in pumello ( Citrus grandis ) enhanced cold tolerance, reduced electrolyte leakage by 13% and increased proline levels by up to 67% compared to wild-type plants 101 .

Flowering time and dormancy release

Flowering time is a very important trait targeted for improvement in fruit crops because of its close association with the timing of fruit development. This trait is under strict genetic regulation and is dependent on environmental conditions, particularly temperature and day length, which limits the geographical regions in which crops can be cultivated 102 . Genetic engineering has been used to express floral activators or repressors, allowing the specification of floral transition and dormancy requirements in major fruit tree species. In transgenic apple, plum, and citrus trees, the overexpression of FT family floral activators needed to trigger bud breaking promoted early flowering (by up to 45 weeks in apple and 12 weeks in orange), and reduced dormancy requirements, eliminating them completely in plum 103 , 104 , 105 . Recently, CRISPR/Cas9 was used to inactivate the self-pruning 5G gene in tomato, which abolished sensitivity to day length and reduced the time to harvest by 2 weeks, translating to a greatly accelerated flowering stage and early fruit yield 102 . In kiwifruit, CRISPR/Cas9-mediated repression of the CEN-like genes also led to rapid and early terminal flowering 106 . These experiments provide insights into the genetic and environmental control of flowering time in different fruits and form the basis for additional engineering strategies to develop early or late-flowering cultivars adapted to specific growing regions.

Fruit ripening and sensory attributes

The modulation of fruit ripening is one of the major strategies by which flavor, aroma, and nutrient profiles can be adjusted, and by which the shelf-life can be extended to improve marketability and reduce waste. In climacteric fruits such as apple, banana, and tomato, the key targets are genes associated with ethylene biosynthesis and degradation. In apple, the silencing of ACS and ACC oxidase ( ACO ) by expressing antisense RNA generated fruit that produced 60% less ethylene, increasing firmness by 20% and allowing cold storage for up to 3 years 107 . Although the synthesis of volatile esters was suppressed, sugar and organic acid accumulation were unaffected. Co-suppression and knockdown of ethylene-biosynthetic genes achieved similar results in pear, kiwifruit, and papaya 108 , 109 , 110 .

Sugar and organic acid content can be modified to enhance fruit flavor. In strawberry, the suppression of ADP-glucose pyrophosphorylase by expressing antisense RNA under the control of a fruit-specific promoter inhibited the conversion of sugar to starch and reduced the starch content of transgenic fruits by up to 47% while increasing the soluble sugar content by up to 37% 111 . Plant pigments such as anthocyanins and carotenoids are also major targets for metabolic engineering in fruits because they provide health benefits and allow the production of fruits with unique colors. The overexpression of MYB family transcription factors in apple, grapevine, and strawberry enhanced the production and storage of anthocyanins, with transgenic fruits accumulating up to 50% more than normal 36 , 112 , 113 . The accumulation of carotenoids has been achieved by the RNAi-mediated silencing of β-carotene hydroxylase in sweet orange, preventing conversion of β-carotene to xanthophylls and thus increasing the β-carotene content in the fruit pulp by 26-fold. Caenorhabditis elegans adults fed with diets supplemented with β-carotene-enriched orange pulp were 20% more resistant to hydrogen peroxide-induced oxidative stress than those fed with control diet 114 . These studies demonstrate how genetic engineering and genome editing can be used to produce fruits with enhanced flavor, texture, and nutrient levels.

Trans-grafting

Grafting is widely used during the propagation of fruit trees to allow the selection of rootstock and scions with different favorable characteristics that may be difficult or laborious to combine in one cultivar (such as high fruit yields paired with resistance to root pests). The rootstock and scion still influence each other by exchanging soluble signals, but the two components maintain their genetic integrity 115 . Trans-grafting refers to grafting of a non-transgenic scion onto a transgenic rootstock. Some desirable characteristics of the rootstock, such as dwarfing or disease resistance, are conferred upon the scion by the vascular transport of RNA, hormones or signaling proteins, but the shoot, leaves, and fruits remain transgene-free 116 , 117 . Although the specific regulations vary by country, trans-grafting can be used to circumvent restrictions on the marketing of GM products in certain jurisdictions 118 . This technology has been used in apple, by grafting non-transgenic scions onto rootstock expressing the Agrobacterium rhizogenes rolB gene, which confers dwarfing characteristics on the scion 119 . In grapevine, non-transgenic scions were grafted onto rootstocks engineered to produce an antimicrobial peptide and a protein that inhibits cell wall degradation. These proteins were transported to the scion through the xylem, resulting in the enhanced mobilization of water and nutrients and a 30–95% reduction in pathogen-induced mortality 120 . Transgenic rootstocks can therefore improve the production of commercially important fruit trees but the fruits and seeds do not carry any exogenous DNA 79 .

Moving beyond transgenesis—genome editing technologies

Genome editing is perhaps the most important recent development in crop breeding, and protocols based on the versatile CRISPR/Cas9 system have been optimized for several fruit species to increase the editing efficiency. In apple, CRISPR/Cas9 produced transgene-free edits 121 . In cucumber, wild strawberry, and watermelon, CRISPR/Cas9 constructs were integrated as part of the T-DNA but segregation was then achieved through back-crossing 122 , 123 , 124 , 125 . A major challenge to the commercial development of edited varieties is the successful transmission of targeted mutations through the germline 126 . This is particularly difficult in woody species, including fruit trees, because they are propagated vegetatively. Back-crossing could take decades (depending on the species) and could result in the unintentional outcrossing of the edited gene. It is also difficult to achieve homozygosity at the edited locus within the desired genetic background because most fruit trees are self-incompatible and thus require obligate outcrossing. Such characteristics hinder the introduction of genome edits that are stable and heritable 127 , 128 , 129 . Several new derivatives of the original CRISPR/Cas9 editing platform have been proposed, including CRISPR/Cas9 ribonucleoprotein (RNP) technology, CRISPR cytidine and adenosine base editors (CBEs/ABEs), CRISPR flippase, and new CRISPR-associated nucleases such as Cas12a/Cpf1, which may help to address these challenges and accelerate the development and commercialization of genome-edited crops 77 , 126 , 129 , 130 , 131 , 132 .

CRISPR RNP technology

Transgene-free genome editing improves the commercialization potential of modified crops (including fruits) because the CRISPR/Cas9 cassette is not inserted into the genome and, in many jurisdictions, the resulting variety is regulated in the same manner as a conventional crop, with certain caveats 21 . CRISPR/Cas9 RNP technology avoids transgene integration by delivering purified RNPs containing the Cas9 protein and gRNA into plant protoplasts and the subsequent regeneration of plants 133 , 134 . This approach has already been used in apple and grapevine to introduce mutations that confer resistance to fire blight and powdery mildew, respectively 129 . In addition to Cas9 RNPs, CRISPR/Cpf1-RNPs have also been employed successfully for gene editing in protoplasts of soybean and tobacco, paving the way for future use in other crops, including fruits and vegetables 134 . Subsequent optimization experiments permitted plant regeneration from protoplasts and improved the transformation protocol for grape protoplasts, reducing the amount of time needed for RNP delivery and genome editing to less than 3 weeks 131 . It is likely that species- and even cultivar-specific protocol optimization will be necessary to achieve satisfactory editing efficiencies because the major hurdle is not the delivery of RNPs across the protoplast membrane, but the subsequent recovery and regeneration of fertile plants.

CRISPR base editing

Whereas conventional CRISPR/Cas9 editing tends to introduce short insertions or deletions at the target locus, cytidine and adenosine base editing facilitates the targeted introduction of single nucleotide replacements by direct C-to-T or A-to-G base conversion, respectively. Base editors have been used to introduce herbicide resistance traits in fruit crops in proof-of-concept experiments. For example, CBE in the watermelon ALS gene resulted in a single amino acid substitution that was sufficient to confer broad-spectrum and heritable resistance to commercial sulfonylurea herbicides 122 .

CRISPR flippase

Flp/ FRT is a yeast site-specific recombinase system in which the recombinase Flp (flippase) catalyzes recombination between two copies of the 34-bp FRT site, resulting in the excision or inversion of the intervening DNA, depending on the relative orientation of the FRT sites. The Flp/ FRT system has been used to remove selectable markers in T1 apple, apricot, citrus, and grapevine plants, leaving a single FRT site behind as a footprint 2 . These studies laid the foundations for more recent work in which the FLP/FRT system was placed under the control of a heat-shock promoter and incorporated into the CRISPR/Cas9 plasmid, allowing the editing of a disease susceptibility gene in apple and subsequent removal of the CRISPR/Cas9 components 77 . This technology has yet to be applied in other fruit crops, but it shows great promise given the efficiency of editing and T-DNA excision.

New CRISPR nucleases

Most CRISPR studies thus far have used the endonuclease Cas9 from Streptococcus pyrogenes (SpCas9). In its native form, SpCas9 requires a trans-activating CRISPR RNA (tracrRNA) and a CRISPR-RNA (crRNA) to induce blunt double-strand breaks in target DNA. These functions were combined into a single gRNA for the development of CRISPR/Cas9 as an engineering tool. But SpCas9 is only one of a large family of CRISPR-associated nucleases with diverse properties, some of which may be advantageous for genome editing in fruit crops by improving efficiency, specificity, or versatility, or by reducing costs 135 . For example, Cas9 from Staphylococcus aureus (SaCas9) differs from SpCas9 in terms of protospacer adjacent motif (PAM) specificity but has a similar editing efficiency. It has been used in several model plant species and also recently in citrus, and provides greater versatility by extending the range of potential genomic targets 126 .

Cas12a/Cpf1 from Prevotella and Francisella spp. recognizes a T-rich PAM and generates compatible cohesive ends with overhangs of 4–5 nt, differing from the blunt ends introduced by Cas9, and increasing the efficiency of DNA integration (knock-in) 136 . Cas12a/Cpf1 is also a smaller protein than Cas9, which improves the efficiency of multiplex editing. CsmI is also smaller than Cas9 136 , and recognizes AT-rich PAM sites thus improving the accuracy of genome editing in AT-rich regions 135 . This approach has been employed to edit the PDS gene in citrus, establishing the feasibility of Cpf1-mediated, DNA-free editing in fruit crops 137 .

Conclusions

Genetic engineering facilitates the development of fruits with useful agronomic or quality traits that are difficult or laborious to achieve by conventional breeding, either due to the lack of suitable germplasm or the long breeding cycles and need for multiple rounds of back-crossing. The same traits can be introduced by genetic engineering in one generation, often directly into elite varieties. Some genetically engineered fruits have been on the market for more than 25 years, and have achieved a remarkable positive socioeconomic impact by reducing pests and diseases and increasing the quality of the end product, both of which help to increase income for farmers. Further benefits to farmers, consumers, and the environment reflect the reduced use of pesticides. The development of new molecular breeding technologies such as trans-grafting and genome editing not only offer the promise of further commercial fruit varieties with resistance to biotic and abiotic stresses, improved flavor and nutrient content, and modified flowering and ripening times, but also help to address some of the regulatory constraints that limit the cultivation of first-generation transgenic crops. In particular, the development of transgene-free genome editing methods based on CRISPR/Cas9 and other nucleases offers a way to introduce precise changes at preselected genomic sites with no genetic footprints and no off-targets. In many jurisdictions, some varieties generated through genome editing are exempt from GMO regulations. These tools and techniques are available for the accelerated development of fruit crops with properties that satisfy the needs of producers, retailers, and consumers, in a sustainable and environmentally friendly manner.

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Acknowledgements

M.L.-G., P.S.G.-C., P.C., and T.C. would like to acknowledge funding from MINECO, Spain (PGC2018-097655-B-I00 to P Christou), Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit (ABBU). P.S.G.-C. was supported through an Agrotecnio postdoctoral fellowship. A.D. acknowledges the support from Washington State University Agriculture Center Research Hatch grant WNP00011.

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P.C and A.D. conceived the idea. M.L.-G. and P.S.G.-C. planned the outline. M.L.-G., S.L.H., and P.S.G.-C. collected the literature and wrote the paper. M.L.-G. and P.S.G.-C. prepared the figure and tables. P.C., T.C., and A.D. critically reviewed and improved the paper. All authors approved the final version.

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Lobato-Gómez, M., Hewitt, S., Capell, T. et al. Transgenic and genome-edited fruits: background, constraints, benefits, and commercial opportunities. Hortic Res 8 , 166 (2021). https://doi.org/10.1038/s41438-021-00601-3

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The Protection of Plant Varieties and Farmers’ Rights Act of India

7 Pages Posted: 31 Dec 2020

Saravana Kumar

Agricultural College and Research Institute

Date Written: October 28, 2020

The Protection of Plant Varieties and Farmer's Rights Act, 2001 (PPVFR Act 2001) was enacted by India as a part of sui generis system. The Act has 11 chapters and 97 clauses. For a variety to be eligible for registration, it must conform to the criteria of novelty, distinctiveness, uniformity and stability (NDUS) under Section 15 of the act. After the variety had been registered the breeder or the farmer had the right to produce, to sell, to market, to distribute, to import and to export the variety. The period of protection is eighteen years in the case of trees and vines and fifteen years in the case of extant varieties. It also provides equitable benefit sharing. Thus the Indian PVPFR Act provides a balance between plant breeders’ rights along with farmers’ rights and researchers’ rights and thus appears to be an effective sui generis system.

Keywords: Breeders’ Right, Distinct, Extant Variety, Farmers’ Right, Novelty

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Saravana Kumar (Contact Author)

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Eachangkottai Tamil Nadu Agricultural University Thanjavur India

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The synthesis of papaya fruit flavor-related linalool was regulated by CpTPS18 and CpNAC56

• Original Article
• Published: 15 November 2023

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• Yuan Yao 1   nAff2 ,
• Wenhui Fu 3 ,
• Suyan Wan 1 ,
• Wenping Zhang 4 &
• Ray Ming   ORCID: orcid.org/0000-0002-9417-5789 1  

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Papaya is a tropical fruit crop renowned for its rich nutrition, particularly pro-vitamin A. Aroma substances are a major component of fruit quality. While extensive research has been conducted on papaya aroma, there has been a notable lack of in-depth research into a specific class of substances. To bridge this gap, our study focused on analyzing the aroma components of various papaya varieties and their biosynthesis pathways. We compared the volatile components of three papaya varieties with distinct flavors at various ripeness stages. A continuous accumulation of linalool, a volatile compound, in the 'AU9' fruit was detected as it matured. The linalool content reached 56% of the total volatile components upon full ripening. Notably, this percentage was significantly higher than that observed in the other two varieties, ‘ZhongBai’ and ‘Malaysian 7’, indicating that linalool serves as the primary component influencing the papaya's odor. Subsequently, we identified CpTPS18 , a gene associated with linalool biosynthesis, and demonstrated its ability to catalyze linalool production from GPP and enhance its accumulation through overexpression in papaya fruits, both in vivo and in vitro. Based on transcriptomic analysis, it was predicted that CpMYB56 and CpNAC56 may transcriptionally activate the expression of CpTPS18 . Subsequent yeast one-hybrid assay and dual luciferase analysis revealed that CpNAC56 activates the transcription of CpTPS18 . Transient overexpression in vivo demonstrated that this gene could upregulate the expression of CpTPS18 and promote linalool accumulation. These results uncovered the primary volatile molecule responsible for papaya fruit odor and identified two major genes influencing its biosynthesis. The genomic resources and information obtained from this study will expedite papaya improvement for fruit quality.

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Acknowledgements

This work was supported by the startup fund from Fujian Agriculture and Forestry University.

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Center for Genomics and Biotechnology, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

Yuan Yao, Suyan Wan & Ray Ming

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School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya, 572025, China

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Yao, Y., Fu, W., Yu, Y. et al. The synthesis of papaya fruit flavor-related linalool was regulated by CpTPS18 and CpNAC56 . Plant Reprod (2023). https://doi.org/10.1007/s00497-023-00486-3

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Phyllosphere-associated microbes can significantly alter host plant fitness, with distinct functions provided by bacteria inhabiting the epiphytic (external surface) vs. endophytic niches (internal leaf tissue). Hence, it is important to understand the assembly and stability of these phyllosphere communities, especially in field conditions. Broadly, epiphytic communities should encounter more environmental changes and immigration, whereas endophytic microbiomes should face stronger host selection. We analysed the structure and stability of leaf phyllosphere microbiomes of four traditionally cultivated rice landraces and one commercial variety from northeast India grown in the field for three consecutive years, supplemented with opportunistic sampling of 8 other landraces. Epiphytic and endophytic bacterial communities shared dominant core genera such as Methylobacterium and Sphingomonas. Consistent with an overall strong environmental effect, both communities varied more across sampling years than across host landraces. Seeds sampled from a focal landrace did not support vertical transmission of phyllosphere bacteria, suggesting that both types of communities are assembled anew each generation. Despite these points of convergence, epiphytic communities had distinct composition and significantly higher microbial load, and were more rich, diverse, modular, and unstable than endophytic communities. Finally, focused sampling of one landrace across developmental stages showed that the divergence between the two types of communities arose primarily at the flowering stage. Thus, our results show both convergent and divergent patterns of community assembly and composition in distinct phyllosphere niches in rice, identifying key bacterial genera and host developmental stages that may aid agricultural interventions to increase rice yield.

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Effects of different fertilization rates on growth, yield, quality and partial factor productivity of tomato under non-pressure gravity irrigation

Qing-jie du.

College of Horticulture, Henan Agricultural University, Zhengzhou, Henan, China

Huai-Juan Xiao

Jia-xin zhang, lu-yao zhou, ji-qing wang, associated data.

All relevant data are within the manuscript and its Supporting Information files.

To select the optimum fertilizer application under specific irrigation levels and to provide a reliable fertigation system for tomato plants, an experiment was conducted by using a microporous membrane for water-fertilizer integration under non-pressure gravity. A compound fertilizer (N:P 2 O 5 :K 2 O, 18:7:20) was adopted for topdressing at four levels, 1290 kg/ha, 1140 kg/ha, 990 kg/ha, and 840 kg/ha, and the locally recommended level of 1875 kg/ha was used as the control to explore the effects of different fertilizer application rates on growth, nutrient distribution, quality, yield, and partial factor of productivity (PFP) in tomato. The new regime of microporous membrane water-fertilizer integration under non-pressure gravity irrigation reduced the fertilizer application rate while promoting plant growth in the early and intermediate stages. Except for the 990 kg/ha fertilizer treatment, yields per plant and per plot for each fertilizer application rate were higher than or equal to those of the control. The new regime could effectively improve PFP and reduce soil nutrient enrichment. Fertilizer at 840 kg/ha showed the optimum results by increasing PFP by 75.72% as compared to control. In conclusion, the fertilizer rate at 840 kg/ha has not only maintained the productivity of soil but also tomato growth and quality of fruit which makes the non-pressure gravity irrigation a potential and cost-effective way for fertilizer application.

Introduction

Fertilizers, which are indispensable and the most important material input in modern agricultural production [ 1 , 2 ], have played a vital role in improving the yield and quality of crops [ 3 – 5 ]. However, over-fertilization not only inhibits the improvement of crop yield and quality, it also results in serious issues, such as hardening and acidification of the soil, aggravation of crop pests, leaching loss of soil nutrients, and threats to groundwater safety. These issues exert a serious impact on agricultural sustainability and the ecological environment [ 6 – 8 ]. In recent years, integrated techniques of water and fertilizer application have been widely developed and popularized. These studies have conducted irrigation and fertilization trials according to the soil nutrient status and the crop water and fertilizer requirements, thus achieving the purpose of saving water and fertilizer, increasing production and quality, and protecting the ecological environment [ 9 – 11 ]. The microporous membrane water-fertilizer integration technique is newly developed. By adopting microporous membranes as a substitute for drip irrigation tapes and pipes, which involves placing a perforated membrane in the furrow between two cultivation ridges and covering it with a plastic film, water can flow between the plastic film and the perforated membrane and infiltrate into the soil through pores on the membrane under non-pressure gravity irrigation. This technique not only saves irrigation equipment costs, but it also eradicates blocking, and has better irrigation uniformity [ 12 ].

To date, numerous studies have explored the effects of fertilizer application rate on crop growth, yield, and quality [ 4 , 9 , 13 ]. For example, Qu et al. [ 14 ] found that the yield increased with a rising fertilizer application rate up to a point, after which yield decreased in cucumbers grown in substrate bags in spring. Zhang et al. [ 15 ] noticed that, compared to the conventional fertilizer application method, a controlled-release fertilizer management method significantly increased yield, with more accumulated total dry weight in bitter gourd. Currently, the techniques integrating microporous membrane water and fertilizer application are largely based on drip irrigation tapes, and the possibility of non-pressure gravity irrigation by water-fertilizer integration using microporous membranes has rarely been explored.

In this research, we employed the integrated microporous membrane water and fertilizer technique for non-pressure gravity irrigation, to determine the optimal fertilizer application rate of spring tomato cultivated in a plastic greenhouse under a specific irrigation level and to clarify the fertilizer requirements of tomato, to thus provide a theoretical basis for efficient fertilizer application and cultivation.

Material and methods

Experimental material, site, and time.

The ‘K1602’ tomato variety was employed in this study. Water-soluble fertilizer (N:P 2 O 5 :K 2 O, 18:7:20) and base fertilizer (N:P 2 O 5 :K 2 O, 15:15:15) were obtained from Yichuan Fufeng Plant Nutrients & Fertilizers Co., Ltd. The perforated plastic film was 60 cm wide and 6.4 m long. Holes (3-mm diameter) in the film were spaced at intervals of 20 cm lengthwise and 12.5 cm across, with three holes punched in each row. The experiment was performed at Zhengzhou Zhengyan International Seed Technology Exhibition Park (Xinzheng, China) (34°16′ to 34°39′ N, 113°30′ to 113°54′ E), from March 16 to July 12, 2017.

Experimental design and treatment

Four different fertilizer levels were examined in this experiment: 1290 kg/ha (FA), 1140 kg/ha (FB), 990 kg/ha (FC), and 840 kg/ha (FD), respectively. The local traditional fertilizer application level (1875 kg/ha) was set as the control (CK). The irrigation amount and target yield of all treatments were 1650 m 3 /ha and 11.25 t/ha, respectively.

Tomato seedlings were planted at the six-leaf stage. Each plot area was 7.8 m 2 (6 m × 1.3 m), with three biological replicates. The base fertilizer was applied in the form of dried chicken manure (1.5 × 10 4 kg/ha) and compound fertilizer (N:P 2 O 5 :K 2 O, 15:15:15, 525 kg/ha). The CK treatment used furrow irrigation to fertilize plants, and integrated irrigation and fertilizer application was performed in other treatments. The times and amounts of irrigation and fertilization are listed in Table 1 .

Measurements of crop parameters

For analysis, seven plants were randomly selected from each treatment from one replicate. Plant height, stem diameter, and leaf number were measured 20, 40, and 60 d after planting. Fresh and dry weight as well as the nitrogen (N), phosphorus (P), and potassium (K) contents of plants were measured after uprooting. Soil samples before transplanting and after uprooting were collected from the 0–20, 20–40, and 40–60 cm soil layers using a five-point sampling method [ 16 ]. The third truss fruits were picked to determine fruit quality.

Plant height was measured using a tape measure, while basal stem diameter (at the midpoint between the stem base and cotyledon) was measured with Vernier calipers. After fresh weights of roots, stems, and leaves were measured, samples were dried at 105°C for 15 min, and then dried at 85°C until reaching a constant mass. Leaf number was counted on plants (those less than 5 cm in length were excluded). The N, P, and K contents were measured using the Kjeldahl method, vanado-molybdate colorimetry, and flame photometry, respectively [ 17 , 18 ]. The contents of soluble sugars, soluble proteins, vitamin C, soluble solids, and organic acids were measured according to the methods of Rahi et al. [ 19 ] and Tudor-Radu et al. [ 20 ]. The lycopene content was calculated according to the method of Kumar et al. [ 21 ], while the fruit yield was measured for each plot. Partial factor of productivity (PFP) was calculated as

where Y (kg/ha) is total yield of crop fruit and I (kg/ha) is the total fertilizer application amount throughout the growth period.

Data analysis

All data are presented as the mean ± standard error (SE) of three replicates and were analyzed using Data Processing Software (DPS, version 7.05) following one-way analysis of variance (ANOVA). Significant differences ( P < 0.05) among treatment means after controlling for multiple comparisons were determined from a least significant difference (LSD) test.

Effects of different fertilizer application rates on plant growth at different periods

Fertilizer application rate affected plant growth ( Table 2 ). At the 20 th d after planting, plant height and stem diameter of FB and FC plants and leaf number of FA and FC plants, were significantly higher than those of CK plants. By the 40 th d, plant height of FA and FB plants, stem diameter of FC plants, and leaf number of FA plants were respectively increased by 17.70%, 17.04%, 15.54%, and 8.14%, compared to CK plants. By the 60 th d, there were no significant differences among various fertilizer treatments in plant height, stem diameter, or leaf number. These results indicated that fertilizer mainly had an effect during the early and intermediate stages of plant growth, among which the effect of the FC treatment was best.

Data are means ±SE. Different letters within the same column denote statistically significant differences between treatments (P<0.05). The same below.

Effects of different fertilizer application rates on fresh and dry weight

Fresh and dry weight of roots under FA treatment were highest, which were 37.31% and 47.00% higher than those under the CK treatment, respectively ( Table 3 ). The fresh weight of stems was not significantly different among the FB, FC, and CK treatments, but significantly greater than that in the FD treatment. The dry weight of stems was not significantly different among the FB, FC, FD, and CK treatments, which were each significantly lower than that in the FA treatment. Fresh and dry weights of leaves increased as fertilizer application amount decreased except for the FD treatment; those of the FA, FB, and FC treatments were significantly higher than those in the CK treatment, with the FC treatment having the highest values, 97.37% and 95.69% higher than those in the CK treatment, respectively.

Effects of different fertilizer application rates on N, P, and K contents of tomato plants

Total N content in the roots was not significantly different among the FC, FD, and CK treatments and significantly greater than that in the FA and FB treatments ( Table 4 ). Total N content in the stems and leaves were lowest under the FD and FC treatments, respectively, which were not significantly different compared with the CK treatment. The P content of the roots under the FC treatment was higher than that in the CK treatment; no significant differences in P content of the roots or stems were recorded between any other fertilizer treatment and the CK treatment. P content of the leaves under the FA treatment was higher than in the CK treatment, while that in the other fertilizer application treatments was lower than that in the CK treatment. The K content of the roots and stems were greatest under the FD and FC treatments, respectively, and significantly greater than that in the CK treatment. However, the K content of leaves in the FC and FD treatments were lower than in the CK treatment.

Effects of different fertilizer application rates on N, P, and K contents in different soil layers

To analyze the nutrient surpluses in the soil, the N, P, and K contents of soil before planting and after uprooting in different layers were measured ( Table 5 ). The N content of the CK treatment in the top soil layer (0–20 cm) was not significantly different from the soil before transplanting, while the same layers of FA and CK soil were greatly higher in N content than those of the FC and FD treatments. The P content of the soil before transplanting was sharply lower than the FA and FC soil, and that no significant differences were found among the FB, FD, and CK treatments. For K content, there were no significant differences among fertilizer levels, and that of each treatment was significantly lower than in the soil before transplanting. In the 20–40 cm soil layer, the N contents of the CK treatment was remarkably greater than in the soil before transplanting and other fertilizer treatments. The P content in CK soil was significantly lower than that in the soil before transplanting and FA soil and sharply higher than in the soil in the other treatments. Except for the FB treatment soil, the K content of CK soil was not notably different from the other treatments. In the drip soil layer (40–60 cm), the N content of FC and FD soils were significantly lower than that of CK soil. The P content of CK soil was highest, while that of FC soil was lowest. The K content of CK soil was significantly lower than of FA and FB soils and not different from that of basic soil or the other fertilizer levels. Thus, N, P, and K enrichment under the FC and FD treatment was decreased in drip soil.

Effects of different fertilizer application rates on tomato quality

Soluble sugar content under the FD treatment was highest, 2.4% higher than that under the FC and CK treatments ( Table 6 ). There was no significant difference in organic acid content under the CK, FC, and FD treatments. The sugar-acid ratio of under the FA, FB, and FD treatments were markedly greater than that of the CK treatment (16.57%, 17.71%, and 11.14% higher, respectively), while those of the FD and FC treatments were not notably different. The lycopene content under the FB, FC, and FD treatments were all significantly higher than that under the CK treatment, with that of the FC treatment being the highest, 39.58% higher than that of the CK treatment. The soluble protein content of the CK treatment was significantly lower than that of the FA treatment (by 8.19%), but greater than that of the other fertilizer treatments. The soluble solid content under the CK treatment was highest, but not significantly different than that under the FA, FC, and FD treatments. The vitamin C content under the FA treatment was greatest, but that was not distinctly different from that of the CK treatment. On the whole, fertilizer treatments FC and FD were more effective in improving tomato fruit quality.

Effects of different fertilizer application rates on yield and PFP

The FA yield per plant was highest, being significantly higher than that of the CK treatment (by 8.82%), while those of the other fertilizer treatments were not significantly different from that of the CK treatment ( Table 7 ). Plot yield for the FA and FB treatments were significantly higher than that under the CK treatment (5.65% and 6.89%, respectively). PFP under the four fertilizer levels were significantly (39.71%, 54.08%, 47.83%, and 75.72%, respectively) greater than that under the CK treatment, while the FD treatment had the highest PFP, with a plot yield that was not significantly different from that of the CK treatment.

Compared to the use of furrow irrigation to apply fertilizer, the microporous membrane water-fertilizer integration technique (which functions like drip irrigation technology) was able to reduce topdressing amounts and promote normal growth in tomato plants. The drip irrigation equipment needs a large initial investment, however, the microporous membrane used in this study was made from a used shed film, which is more economic for growers than installing a drip irrigation system.

Scientific fertilizer application offers an important means of improving crop growth, protecting the ecological environment, and maintaining agricultural sustainability. Plant fresh and dry weight, which reflect the accumulation of plant biomass to some extent, constitute important indicators of growth vigor [ 15 , 22 ]. In general in this study, under the same irrigation conditions, plant growth in the early and intermediate stages increased with decreasing fertilizer application up to a point, after which they decreased. Fertilizer application improved availability of NPK in root zone, leading to an increase in uptake of nutrients to the plant. Many studies have demonstrated this increment in nutrients contributed to plant biomass accumulation is due to higher leaf photosynthetic capacity [ 1 , 23 ]. However, excess fertilizer application would result in low water availability to plant due to high osmotic conditions in soil, and therefore stunted plants [ 24 ]. Our results also showed that the total NPK contents of FC plants were the highest, corresponding to the best plant growth and highest dry weight.

Nutrient content in the surface soil is influenced by fertilizer application rate, irrigation, and plant species, as nutrients are partially taken up by plants and partially migrate downward deep into soil as moisture moves [ 25 , 26 ]. In the present study, N and K were enriched in deep soil, especially under high fertilizer application, but the contents of N and K in 0–20 cm soil were similar or lower than that in soil before treatment. In soil, N and K are mobile nutrients. Displacement of N and K through the soil profile often occur with irrigation during over application [ 27 , 28 ]. This may be responsible for the N and K distribution in soil under high fertilizer application. On the contrary, P enrichment was found in topsoil, which may be due to high Ca 2+ concentrations in water and soil in experimental site [ 29 , 30 ]. In calcareous soils, P can be immobilized into insoluble compound, such as calcium-phosphate minerals, leading to low mobility [ 31 , 32 ]. The high residues of nutrient would increase the risk of groundwater pollution. Thus, the 840 kg/ha fertilizer application rate met the nutrient needs of tomato plants, without leaving excess residues in the soil, making it the optimal fertilizer application rate.

In this study, the PFP under four fertilization levels were significantly higher than that of the CK treatment. This suggested that the microporous membrane water-fertilizer integration technique could improve PFP. Moreover, PFP is greatly affected by fertilizer supply level. We found that the highest PFP occurred under the lowest fertilizer amount (840 kg/ha). Previous studies also confirmed that the highest PFP often occur under a low fertilizer supply level [ 26 , 33 ]. According to fertilizer response function, positive effects of fertilizer on yield may be responsible for the high PFP under low fertilizer supply level [ 34 ]. On the other hand, high fertilizer input increased nutrient leaching losses, and therefore low PFP [ 22 , 35 ].

Fertilizer is an important determinant for yield formation. High fertilizer application under non-pressure gravity irrigation enhanced yield compared with CK while no significant difference in yield was observed between lowest fertilizer treatment (840 kg/ha) and CK. Interestingly, the yield was lowest under 990 kg/ha treatment. This may be attributed to more photosynthate allocation to vegetative growth, thereby impacting their reproduction. Under non-pressure gravity irrigation, the fertilizer application rate was declined by 31.2%-53.6% compared with CK. Thus, the decreased fertilizer supply under non-pressure gravity irrigation have no negative effect on tomato plant growth and yield. Specifically, the 840 kg/ha fertilizer treatment achieved the highest economic value under non-pressure gravity irrigation. Although the per plant and plot yields under 840 kg/ha fertilizer treatment were 8.8% and 5.7% lower than that under the 1290 kg/ha fertilizer treatment, the fertilizer input decreased by 34.8%. Considering the fertilizer input, nutrient residues and yield, 840 kg/ha fertilizer application is recommended.

In conclusion, the 840 kg/ha fertilizer application rate most effectively improved the yield, PFP, and soil productivity maintenance, with better quality of tomato fruits. Our results preliminarily identified a specific irrigation and fertilization regime for tomato cultivation under non-pressure gravity irrigation. In future work, the optimal application proportion of NPK for tomato should be assessed under non-pressure gravity irrigation.

Supporting information

Funding statement.

The work was supported by the Special Support Project for the Construction of Technology Systems for the Bulk Vegetable Industry in Henan Province (CZ2015S003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Wanted: seeds of Dikke Leidsche Winter and other 'lost' heritage varieties

A century ago, there were many more varieties in Dutch agricultural and horticultural sectors than there are today. The Centre for Genetic Resources, the Netherlands (CGN) at Wageningen University & Research is trying to rediscover and preserve ‘lost’ heritage varieties. Through a public action, the CGN hopes to add the last dozens of varieties missing from the list - five of which are ‘most wanted’ - to the collection. “Perhaps someone somewhere still has some seeds of old vegetable varieties in the attic."

Heritage varieties are varieties of crops that were developed and used until about the 1950s, says Robbert van Treuren, researcher on crop genetic resources. "Due to the expansion in agriculture, these varieties have been discarded due to poor yields or limited nutritional value, for example, and have thus fallen into oblivion. But these varieties are part of our biocultural heritage and may possess traits that are useful in today's breeding. Our list includes 350 heritage varieties. We share the stories thereof on our website . The CGN has seed samples of most heritage varieties. These are also available. But material from about 50 varieties is missing. Including the 5 most wanted."

5 most wanted heritage varieties

• Lange Holkruin: This parsnip variety has a large, fleshy white root with a sunken crown. Also known in the Netherlands by the name ‘Utrechtsche Platkop’.
• Wassenaar Selection: Yellow-fleshed cabbage turnip with purple or bronze coloured head and a thin stem. Has a round to oval shape.
• Kennemerland: Grown under flat glass, this Cantaloupe melon was created from a cross with Karbonkel.
• Dikke Leidsche Winter: The hardiest leek from the last century, featuring blue-green and broad leaves with late maturity.
• Delftsche Groene Kortpoot: This small cauliflower can be harvested early. The cabbage is bright yellow in colour, growing on a short stalk.

The most wanted varieties

The list of the 5 most wanted was compiled by researcher Lana de Bruijn. She explains why she wants to highlight these particular 5 varieties. "We would obviously prefer to recover all the lost varieties, but because of their names and place of origin, the most wanted ones capture our imagination the most. I mainly retrieved the information about these varieties from a list compiled by former CGN members, the Orange List. This list includes all varieties grown in the Netherlands between 1850 and 1950. It describes the characteristics of each variety, the region where they were grown and the year or period when they were last seen in the Netherlands. We hope that seeds of the varieties still missing from our collection are still around somewhere."

Importance of biocultural heritage

According to De Bruijn, retrieving and preserving old varieties is important for several reasons. "A heritage variety says something about our history, just like the paintings of Van Gogh and Rembrandt. For example, it provides information about where it was grown, such as what local people enjoyed eating and under what climatic and soil conditions a variety could grow. Take the oat variety ‘Evene’, for example, which we know was grown on very poor soil. And the wheat variety ‘Zeeuwse tarwe’, which did very well in inclement climates. Such traits can, in turn, be useful for breeding more resilient varieties of crops. Apart from their cultural-historical value, heritage varieties therefore also have social value."

Through a large-scale public action, the researchers are trying to find the missing varieties. De Bruijn: “Perhaps someone somewhere still has a few tubes of seeds in the attic that their grandmother once gave them. Or a hobby grower recognises the described features of a crop growing in his kitchen garden. And sometimes, a variety grows wild and is then discovered, as happened with the heritage rye variety, Veluws Kruiprogge." Van Treuren adds: “Or a variety may be found abroad. Perhaps because seeds have been taken when people emigrated, or because some varieties have been grown in several countries, such as the Passe-Partout lettuce variety."

Stories wanted too

Anyone who comes across one or more of the most wanted or other missing varieties can contact the CGN , says De Bruijn. "People can send seeds to us. We will then check whether the quality of the seed is good enough to germinate and grow the variety. Then we will check if it is indeed the variety we were looking for. In case of confirmed identity, we will propagate the seeds and make them available to the public. Seeds of heritage varieties can be ordered through our website or from seed traders." Besides seeds, the CGN is also looking for stories. Van Treuren: “Maybe someone still knows where the seeds came from, or perhaps a recipe using the variety is still present in a drawer somewhere."

Admiring heritage breeds with your own eyes

This year, a dozen heritage varieties are grown in the historic 'Ommuurde Tuin' kitchen garden in Renkum. De Bruijn: “This will literally give people a glimpse into our biocultural heritage and make our collection of heritage varieties a bit more tangible. For example, visitors can see the rather unknown ‘Haverwortel’ growing and flax from which linen is made. It would be nice if through this garden we’re able to reach people who still have seeds of missing varieties, so that we can expand the heritage variety collection."

Centre for Genetic Resources, the Netherlands

The CGN has a collection of nearly 24,000 seed samples of more than 30 different agricultural and horticultural crops. These are kept in vacuum-packed bags in freezers at -20⁰C, with the aim of preserving them for ever. These seed samples are made available to professionals in breeding, research and education. Seed samples of heritage varieties are also accessible to the general public as a special collection. Apart from plants, the CGN also manages a gene bank for animals, trees and aquaculture.

The rate of net assimilation and the rate of relative growth of amaranth (Amaranthus tricolor L.) varieties on urea fertilizer application

• Wulansari, FC
• Purwanto, E.

Nutrient availability affects plant growth and productivity measures, including net assimilation and relative growth rate. Nitrogen, such as amaranth, is vital for enhancing plant growth. Amaranth cultivation requires more nitrogen during its growth, which is met through fertilization. The most commonly used nitrogen fertilizer is urea. The goal of this research activity was to learn whether urea fertilizer had an impact on the relative growth rate and net assimilation rate of amaranth. This research was conducted in Sukosari village, Jumantono, Karanganyar, from July to August 2023. The experimental design employed a completely randomized approach, using two factors: the dose of urea fertilizer (0, 50, 100, 150, 200, 250, and 300 kg.ha -1 ) and the varieties of amaranth (green and red). At a significance level of 5%, the data analyzed variance (ANOVA) and the Duncan Multiple Range Test (DMRT). According to the conclusions, there was no interaction between the urea dose and the type of amaranth. At 300 kg.ha -1 of urea, the plant's height, leaf area, and leaf count all reached their highest points. At a urea dose of 250 kg ha -1 , the rates of dry weight gain, net assimilation, and relative growth were greatest. The net assimilation rate is positively correlated to the relative growth rate but not to other growth parameters.

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Open Access

Peer-reviewed

Research Article

Pharmacological and behavioral investigation of putative self-medicative plants in Budongo chimpanzee diets

Contributed equally to this work with: Elodie Freymann, Fabien Schultz

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected] (EF); [email protected] (FS)

Affiliation Primate Models for Behavioural Evolution Lab, Institute of Human Sciences, Department of Anthropology and Museum Ethnography, University of Oxford, Oxford, United Kingdom

Roles Supervision, Writing – review & editing

Affiliations Primate Models for Behavioural Evolution Lab, Institute of Human Sciences, Department of Anthropology and Museum Ethnography, University of Oxford, Oxford, United Kingdom, Gorongosa National Park, Sofala, Mozambique, Interdisciplinary Centre for Archaeology and the Evolution of Human Behaviour, University of Algarve, Faro, Portugal

Roles Funding acquisition, Supervision, Writing – review & editing

Affiliations Ethnopharmacology & Zoopharmacognosy Research Group, Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany, ZELT–Center for Nutrition and Food Technology gGmbH

Roles Formal analysis, Writing – original draft, Writing – review & editing

Affiliation Ethnopharmacology & Zoopharmacognosy Research Group, Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany

Roles Resources, Supervision, Writing – review & editing

Affiliations Wild Minds Lab, School of Psychology and Neuroscience, University of St Andrews, St Andrews, United Kingdom, Budongo Conservation Field Station, Masindi, Uganda

Affiliation Wildlife Research Center, Inuyama Campus, Kyoto University, Inuyama, Japan

Roles Investigation

Affiliation Budongo Conservation Field Station, Masindi, Uganda

Roles Formal analysis

Affiliations Budongo Conservation Field Station, Masindi, Uganda, Czech University of Life Sciences Prague, Prague, Czech Republic

Roles Resources, Writing – review & editing

Affiliations Budongo Conservation Field Station, Masindi, Uganda, Department of Comparative Cognition, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing

Affiliations Ethnopharmacology & Zoopharmacognosy Research Group, Department of Agriculture and Food Sciences, Neubrandenburg University of Applied Sciences, Neubrandenburg, Germany, Pharmacognosy and Phytotherapy, School of Pharmacy, University College of London, London, United Kingdom

• Elodie Freymann, 
• Susana Carvalho, 
• Leif A. Garbe, 
• Dinda Dwi Ghazhelia, 
• Catherine Hobaiter, 
• Michael A. Huffman, 
• Geresomu Muhumuza, 
• Lena Schulz, 
• Daniel Sempebwa, 

• Published: June 20, 2024
• https://doi.org/10.1371/journal.pone.0305219
• Reader Comments

Wild chimpanzees consume a variety of plants to meet their dietary needs and maintain wellbeing. While some plants have obvious value, others are nutritionally poor and/or contain bioactive toxins which make ingestion costly. In some cases, these nutrient-poor resources are speculated to be medicinal, thought to help individuals combat illness. In this study, we observed two habituated chimpanzee communities living in the Budongo Forest, Uganda, and collected 17 botanical samples associated with putative self-medication behaviors (e.g., bark feeding, dead wood eating, and pith-stripping) or events (e.g., when consumer had elevated parasite load, abnormal urinalysis, or injury). In total, we selected plant parts from 13 species (nine trees and four herbaceous plants). Three extracts of different polarities were produced from each sample using n -hexane, ethyl acetate, and methanol/water (9/1, v/v ) and introduced to antibacterial and anti-inflammatory in vitro models. Extracts were evaluated for growth inhibition against a panel of multidrug-resistant clinical isolates of bacteria, including ESKAPE strains and cyclooxygenase-2 (COX-2) inhibition activity. Pharmacological results suggest that Budongo chimpanzees consume several species with potent medicinal properties. In the antibacterial library screen, 45 out of 53 extracts (88%) exhibited ≥40% inhibition at a concentration of 256 μg/mL. Of these active extracts, 41 (91%) showed activity at ≤256μg/mL in subsequent dose-response antibacterial experiments. The strongest antibacterial activity was achieved by the n- hexane extract of Alstonia boonei dead wood against Staphylococcus aureus (IC50: 16 μg/mL; MIC: 32 μg/mL) and Enterococcus faecium (IC50: 16 μg/mL; MIC: >256 μg/mL) and by the methanol-water extract of Khaya anthotheca bark and resin against E . faecium (IC50: 16 μg/mL; MIC: 32 μg/mL) and pathogenic Escherichia coli (IC50: 16 μg/mL; MIC: 256 μg/mL). We observed ingestion of both these species by highly parasitized individuals. K . anthotheca bark and resin were also targeted by individuals with indicators of infection and injuries. All plant species negatively affected growth of E . coli . In the anti-inflammatory COX-2 inhibition library screen, 17 out of 51 tested extracts (33%) showed ≥50% COX-2 inhibition at a concentration of 5 μg/mL. Several extracts also exhibited anti-inflammatory effects in COX-2 dose-response experiments. The K . anthotheca bark and resin methanol-water extract showed the most potent effects (IC50: 0.55 μg/mL), followed by the fern Christella parasitica methanol-water extract (IC50: 0.81 μg/mL). This fern species was consumed by an injured individual, a feeding behavior documented only once before in this population. These results, integrated with associated observations from eight months of behavioral data, provide further evidence for the presence of self-medicative resources in wild chimpanzee diets. This study addresses the challenge of distinguishing preventative medicinal food consumption from therapeutic self-medication by integrating pharmacological, observational, and health monitoring data—an essential interdisciplinary approach for advancing the field of zoopharmacognosy.

Citation: Freymann E, Carvalho S, Garbe LA, Dwi Ghazhelia D, Hobaiter C, Huffman MA, et al. (2024) Pharmacological and behavioral investigation of putative self-medicative plants in Budongo chimpanzee diets. PLoS ONE 19(6): e0305219. https://doi.org/10.1371/journal.pone.0305219

Editor: Armel Jackson Seukep, University of Buea, CAMEROON

Received: January 9, 2024; Accepted: May 25, 2024; Published: June 20, 2024

Copyright: © 2024 Freymann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Funding for this project was granted by the the Clarendon Fund at the University of Oxford (to EF), the British Institute of Eastern Africa (to EF), Keble College at the University of Oxford (to EF), Boise Trust Fund (to EF), German Federal Ministry of Education and Research (13FH026IX5, PI: L-AG and Co-I: FS) (to LAG, FS) and Neubrandenburg University of Applied Sciences (grant # 13310510) (to LAG, FS).

Competing interests: The authors have declared that no competing interests exist.

Introduction

‘Medicinal foods’ refer to resources in the diet that have potential curative value due to the presence of plant secondary metabolites (PSMs) [ 1 , 2 ]. PSMs are compounds that usually occur only in special, differentiated cells [ 3 ] and which help plants defend against predators, pathogens, and competitors [ 4 – 7 ]. PSMs can have a range of functions, including the inhibition of microbial, fungal, and competitor growth [ 8 ]. While some PSMs can be toxic at high doses, these compounds can also promote the health of human and non-human consumers [ 8 – 10 ]. Research suggests 15–25% of primate and other mammalian diets consist of medicinal foods [ 9 , 11 ]. These resources likely play a critical role in animal health-maintenance by passively preventing or reducing the impact of parasitic infections or other pathogens [ 9 – 14 ].

While most animals likely consume foods with medicinal properties as part of their normal diets, fewer species have been shown to engage in therapeutic self-medication. Huffman [ 15 ] defines this type of self-medicative behavior as the active extraction and ingestion, by an ill individual, of medicinal resources with little nutritional value. Instead of an individual passively benefiting from a plant’s medicinal properties through normal feeding, this form of self-medication requires basic awareness of the resource’s healing properties. One of the best-studied animals to engage in this form of self-medication is our closest living relative: the chimpanzee.

Wild chimpanzees ( Pan troglodytes ), across at least sixteen field sites [ 15 ] have demonstrated therapeutic self-medication using two well-established self-medicative behaviors: leaf swallowing [ 16 , 17 ] and bitter-pith chewing [ 18 ]. Leaf swallowing, first reported by Wrangham [ 19 , 20 ] and described by Wrangham & Nishida [ 21 ], involves the careful selection and ingestion of whole, hispid leaves. This behavior was later demonstrated to expel internal parasites (i.e. Oesophagostomum sp. and Bertiella studeri ) from the gut [ 16 , 17 , 22 , 23 ]. The functional mechanism responsible for this anthelminthic effect is considered to be primarily “mechanical” [ 9 ] as, rather than a chemical compound, the leaf’s indigestibility, brought about by the trichomes on its surface—stimulates gut motility in the swallower [ 17 , 23 , 24 ].

The second established behavior is bitter-pith chewing, which involves the stripping of outer bark and leaves from the soft new stem growth of the shrub, Vernonia amygdalina , exposing the inner pith. Individuals chew the pith and ingest only the bitter juices while spitting out the fibers [ 18 , 25 ]. Bitter-pith chewing is considered ‘phytochemical’ self-medication [ 9 ], as its anthelminthic effect appears to be the result of bioactive PSMs [ 26 – 29 ]. This behavior’s medicinal effect was associated with a significant drop in the infection intensity of Oesophagostomum stephanostomum nematodes [ 25 ], suggesting that the bitter compounds directly affect the adult worms. This hypothesis was supported by in vivo studies conducted by Jisaka et al. [ 30 ], demonstrating that extracts from the pith permanently paralyzed adult Schistosome parasites. V . amygdalina is also used to aid gastrointestinal discomfort and other signs of parasitosis in humans and livestock, symptoms also displayed by chimpanzees ingesting the plant’s bitter pith [ 9 , 18 , 25 , 31 ]. The bitter piths of other plant species are reported to be chewed by chimpanzees across field sites but detailed studies on their medicinal properties have yet to be conducted [ 9 ].

Beyond these two established behaviors, not much is known about the phytochemical self-medicative repertoires of wild chimpanzees, although some behaviors associated with the ingestion of specific plant parts or processing techniques have been recommended for further investigation [ 9 , 15 , 32 ]. One of these behaviors is bark feeding, which involves the ingestion of living stem bark and/or cambium [ 33 ], and which has been observed in at least eleven established field sites [ 33 – 43 ]. Bark feeding has been suggested as a medicinal behavior in chimpanzees and other primates, used to aid in the chemical control of intestinal nematode infection and to relieve gastrointestinal upset [ 9 ]. Bark is characteristically highly fibrous, heavily lignified, sometimes toxic, relatively indigestible, and nutrient-poor [ 44 ]. However, the contribution of bark in chimpanzee diets and toward general health is still poorly understood [though see: 45 ]. In this study, the bark of eight species ingested by Budongo chimpanzees ( Scutia myrtina , Cynometra alexandri , Alstonia boonei , Ficus exasperata , Ficus variifolia , Syzygium guineense , Desplatsia dewevrei , Khaya anthotheca) was screened for antibiotic and anti-inflammatory properties, to better understand the function of bark feeding behaviors and the role this behavior may play in the health maintenance of chimpanzees. For the species K . anthotheca , we tested a mixture of bark and congealed resin, which Budongo chimpanzees were observed to particularly target throughout the study period.

Another putative self-medicative behavior is dead wood eating [ 9 , 35 ], which involves the consumption of decomposing cambium from dead trees. To date, the majority of studies examining this behavior in apes have focused on exploring potential mineral and nutritional benefits, rather than investigating pharmacological properties [ 46 – 49 ]. Many of these studies suggest that dead wood is exploited by chimpanzees as a source of sodium in environments where this mineral is otherwise scarce [ 48 , 49 ]. Our study evaluates the pharmacology of two species of dead wood ( A . boonei and Cleistopholis patens) consumed by the Sonso community of chimpanzees to determine whether this behavior may have multiple functions or health benefits.

The ingestion of pith material from other species has also been suggested as putatively self-medicative [ 34 , 50 , 51 ]. However, unlike V . amygdalina bitter-pith, some of these plant piths appear bland or tasteless. While Wrangham et al. have previously suggested that pith is likely a high-fiber fallback food [ 52 ], De la Fuente et al. review several pith species targeted by chimpanzees with proposed medicinal properties [ 32 ]. In our study, two species of non-bitter piths ( Marantachloa leucantha and Acanthus polystachyus) , were collected for pharmacological assessment. M . leucantha was observed on several occasions being stripped, masticated, and spat out after the juice was extracted from the pith, whereas A . polystachyus was observed being stripped, masticated, and swallowed. Both of these species are also ingested by chimpanzees in Kibale National Park, Uganda [ 52 ].

Establishing phytochemical self-medicative behaviors in wild animals is difficult and time consuming, as the burden of proof is high, self-medicative events can be rare relative to other behaviors, and methods often require multidisciplinary expertise and collaboration [ 9 ]. Past studies have utilized ethnopharmacological methods to determine specific medicinal properties of foods consumed by primates [ 11 ], greatly advancing our understanding of the relationship between primate diets and health. However, a key challenge for establishing novel self-medicative behaviors is differentiating between medicinal food consumption and therapeutic self-medication. While pharmacological data interpreted on its own is crucial for establishing the presence of medicinal resources in chimpanzee diets, the integration of observational and health monitoring data is needed to parse therapeutic self-medicative behaviors from normal feeding behaviors with inadvertent health benefits. Furthermore, the importance of collecting in situ samples from the locations where putative self-medicative behaviors are observed is paramount, as ecological, climatic, and anthropogenic variables can cause variation in the bioactivity of plants across habitats [ 53 ].

In total, we investigated the bioactivity of 51 plant extracts produced from 17 part-specific samples (across 13 species), collected in the Budongo Forest. Each extract was tested for inhibition of bacterial growth as well as anti-inflammatory COX-2 inhibition activity. Due to limitations in scope, funding, and the unavailability of anthelminthic assays for wild animal parasites, none were not conducted in this study, restricting specific identification of parasiticidal behaviors. Assay results are reported and contextualized in this study with direct behavioral evidence and health monitoring data.

Materials and method

Study site and subjects.

Behavioral data, health monitoring metrics, and botanical samples were collected from the Budongo Central Forest Reserve in Uganda (1°35′– 1°55′ N, 31°18′–31°42′ E). An overview of methodological workflow can be found in S2 Fig . The Budongo Conservation Field Station (BCFS) site, founded in 1990, is composed of continuous, semi-deciduous forest and contains two habituated Eastern chimpanzee ( Pan troglodytes schweinfurthii ) communities [ 54 ]. The Sonso community has been studied continuously since 1992, and the ages, social relationships, demographics, and diet of its members are well documented [ 55 , 56 ]. The Sonso population was ~68 individuals at the time of data collection, and the home range covered an area of ~5.33 km 2 [ 57 ]. Waibira, a larger group of at least 105 individuals, was more recently habituated, with consistent data collection beginning in 2011. The Waibira maximum home range area was ~10.28 km 2 [ 57 ].

Behavioral data collection

All samples were collected in the Budongo Forest within the Sonso home range, based on behavioral observations from the study period and supporting evidence from the site’s long-term data of their use. Behavioral and health data were collected from two neighboring chimpanzee communities, each for one four-month field season (Sonso: June-October 2021, Waibira: June-October 2022). Data collected between June-September 2021 informed subsequent plant sample collection for pharmacological analysis, which occurred in early September 2021. Behavioral data collected after sample collection provided additional behavioral context for ingestion of these species. Behavioral data were collected between 07:00 and 16:30 in Sonso and between 06:30 and 17:00 in Waibira using day-long focal animal follows sensu Altman et al. [ 58 ]. This data was recorded using Animal Observer (AO) on iPad and ad libitum feeding events were recorded for any unusual feeding behaviors, including but not limited to bark ingestion, dead wood eating, pith stripping, and geophagy. All feeding events were filmed on a Sony Handycam CX250. We prioritized focal follows on individuals with wounds, high or diverse parasite loads identified through on-going monitoring, or known ailments. However, consecutive day follows of priority individuals were not always possible—or were avoided when they might contribute to increased stress in particularly vulnerable individuals. Throughout the study, using this protocol, 27 Sonso individuals (♂:11; ♀:16) and 24 Waibira individuals (♂:14; ♀:10) were observed. Authors collecting behavioral data were blind to pharmacological results during both study periods.

Health monitoring

Individual health data were recorded in both communities, including opportunistic macroscopic and microscopic fecal analysis and urinalysis testing. While anthelminthic assays were not run in this study, parasite load was opportunistically assessed to provide additional health context for each observation. As the presence of certain helminths may impair a host’s immunological response to bacterial, viral, and protozoal pathogens [ 59 ], parasite load can provide a proxy measurement for overall health. Similarly, a reduced immune system and increased stress caused by co-infections could render a host more susceptible to virulent endoparasites [ 60 , 61 ]. When helminths and/or proglottids were found in samples, they were collected and preserved in ethanol for later identification. To quantify parasite loads, fecal samples were analyzed using the McMaster Method [ 9 , 25 , 62 ]. Urinalysis samples were taken opportunistically using multi-reagent Urine Dipstick Test 9-RC for Urotron RL9 to assess the health and physiological status of group members following methods established by Kaur & Huffman [ 63 ]. Urinalysis metrics considered in this study included: leukocytes (LEU) associated with pyuria caused by UTI, balanitis, urethritis, tuberculosis, bladder tumors, viral infections, nephrolithiasis, foreign bodies, exercise, glomerulonephritis, and corticosteroid and cyclophosphamide use; blood (BLO) associated with peroxidase activity of erythrocytes, and UTIs; and ketones (KET) associated with pregnancy, carbohydrate-free diets, starvation, and diabetes [ 64 ]. Test results were interpreted in situ using a colorimetric scale. We considered a result ‘abnormal’ if the colorimetric scale indicated a positive result when the expected result was negative or if the result was outside the specified test parameters according to the manufacturer.

Plant sample selection for bioactivity testing

Plants were selected for pharmacological testing after three months of data collection in the Sonso community. We selected 10 samples (from 9 species) based on direct observations during this period. These observations included individuals targeting plant parts associated with putative self-medicative behaviors (i.e., bark feeding, dead wood eating, pith-stripping) or sick/wounded individuals seeking out unusually consumed resources. We then selected an additional five species, the ingestion of which had not been directly observed, for testing based on their historical inclusion in Sonso chimpanzees’ bark feeding repertoire. GM, who has worked at the field station for over thirty-years, has previously observed bark feeding on each of these selected species. These historic observations enabled collection of bark samples from specific trees known to have been previously stripped. In two cases, leaf samples were collected from tree species that were also selected for bark samples ( S . guineense and F . exasperata) . While neither Sonso nor Waibira chimpanzees have been observed ingesting the leaves of S . guineense , a sample was collected to enable comparison of bioactivity across plant parts. F . exasperata leaves are consumed in both communities; however, we found no behavioral evidence for use in unusual contexts. In some cases, direct observation of an event involving one of the collected species occurred after botanical collection was complete. These post hoc behavioral observations are reported in this paper, although they did not impact sample selection.

Collection of sample material

Plants were collected from the Sonso community home range following best practice procedures [ 65 ], using sustainable harvesting methods [ 66 ]. See S1 File for more information. Voucher accession numbers are reported in Table 3 . Digital images of voucher specimens can be found in S3 Fig . The currently recognized scientific names of each species were confirmed on https://mpns.science.kew.org/ . Plant family assignments were done in accordance with The Angiosperm Phylogeny Group IV guidance [ 67 ].

Ethnobotanical literature review

We conducted a post-hoc ethnomedicinal review of all species collected for this study using Google Scholar, PROTA, and Kokwaro’s ethnomedicinal pharmacopeia [ 68 ]. To search databases, we used scientific names and synonyms for each plant as keywords [ 65 ].

Plant processing and extractions

At Neubrandenburg University of Applied Sciences, samples were ground using a food processor. Extractions were produced using two solvents and a solvent mixture ( n -hexane, ethyl acetate, and methanol/water ( v/v 9/1)), allowing for the selective isolation of components with varying solubilities and polarities. Methanol-water, the solvent with the highest polarity, generally extracts primary plant metabolites (e.g., polar compounds such as proteins, amino acids, and carbohydrates). Nonpolar solvents like n- hexane extract nonpolar compounds like lipids, making n-hexane a preferred solvent for oil or wax extraction. Extractions with each solvent were achieved through double maceration of new material (non-successively). Extraction suspensions were placed on a shaker at 80 rpm at room temperature for minimum 72h, followed by vacuum filtration. Processes were repeated with the leached material. Filtrates were then combined and dried using a vacuum evaporator, labeled, and stored at -20°C until needed for assays.

Sample solution preparation

To create sample solutions, each crude extract was dissolved in DMSO (Carl Roth) at a concentration of 10 mg/mL. To ensure a homogenous solution, samples were mixed with a vortex mixer and, if necessary, treated with sonication at room temperature or up to 55°C for samples with low solubility. Each extract solution was then tested for inhibition of bacterial growth as well as anti-inflammatory COX-2 inhibition activity. Solutions were stored at -20°C when not in use.

Antibacterial susceptibility tests

A. bacterial strains..

For antibacterial assays, eleven multidrug-resistant clinical isolate strains from nine species were used. This process increased the study’s applicability for early-stage drug discovery, specifically relevant to the threat of antimicrobial resistance (AMR). Seven of these strains (from six species) are classified as ESKAPE pathogens, including Enterococcus faecium (DSM 13590), Staphylococcus aureus (DSM 1104; DSM 18827), Klebsiella pneumoniae (DSM 16609), Acinetobacter baumannii (DSM 102929), Pseudomonas aeruginosa (DSM 1117), and Enterobacter cloacae (DSM 30054), meaning they are highly virulent and resistant to antibiotics [ 69 ]. A strain of the foodborne pathogen Escherichia coli (DSM 498) with AMR as well as a non-resistant E . coli strain (DSM 1576) were also included in the study. Although not an ESKAPE pathogen, E . coli is widely known for causing bacterial diarrhea and AMR strains are a major cause of urinary tract infections [ 70 , 71 ]. Strains of Stenotrophomonas maltophilia (DSM 50170) and Salmonella enterica subsp. enterica (DSM 11320) were also tested. More information on specific clinical isolates/strains, their individual resistance profiles, and antibiotics used can be found in the S5 & S6 Tables in S2 File . Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution testing (M100-S23) were followed [ 72 ].

b. Growth inhibition screening and dose-response study.

The broth dilution in vitro methods for bacterial susceptibility assessment have previously been described by Schultz et al. [ 69 ]. The standardized bacterial working cultures were pipetted into sterile 96-well microtiter plates (Greiner Bio-One International, CELLSTAR 655185). Extracts and antibiotic (64–1 μg/mL), vehicle and sterility controls, were then added into respective wells. Initial optical density measurement (600 nm) was performed, accounting for absorbance of extracts. Plates were incubated at 37°C for 18 h, except for A . baumannii which was incubated for 22h in accordance with strain characteristics ( S5 Table in S2 File ) . After incubation, a final optical density reading (600 nm) was conducted. Percent inhibition values were calculated and the IC 50 and MIC values were determined [ 69 , 73 ]. The IC 50 value is defined as the lowest concentration at which an extract showed ≥ 50% inhibition, and the MIC is the lowest concentration at which an extract displayed ≥ 90% inhibition. A total of 51 samples underwent single-dose pre-screening for growth inhibition (in triplicate) at the concentration of 256 μg/mL on eleven pathogens. Samples showing ≥40% growth inhibition were further tested in a dose-response study with two-fold serial dilution at descending concentrations from 256 to 4 μg/mL. The dose-response experiments were done as biological replicates on separate days in triplicate (technical replicates) to validate reproducibility. Positive controls (antibiotics) and negative controls (vehicle control and sterile media control) were always included. Further details on bacteria standardization can be found in S1 File . Information on plate setup for bacterial library screens and dose-response assays can be found in S4 Fig .

COX-2 inhibition assay

Anti-inflammatory assays were assessed using an in vitro COX inhibitor screening assay kit (Cayman Item No: 701080), with modifications previously described in Schultz et al. [ 74 ]. All extracts were first screened in duplicate for inhibition against human recombinant COX-2 at an initial concentration of 50 μg/mL. For extracts exhibiting at least 50% inhibition, the concentration was then lowered to 10 μg/mL, 5 μg/mL, and 2.5 μg/mL. The most active extracts were taken to dose-response experiments for determination of IC 50 values ( Table 5 ). The assay was done in two steps: 1) the COX reaction step in which the prostaglandin H 2 (PG) was produced (which was further reduced to the more stable prostaglandin F 2α by addition of stannous chloride), and 2) an acetyl choline esterase competitive ELISA step to quantify the produced prostaglandin and calculate a potential enzyme inhibition caused by the extracts. The pure compound and selective COX-2 inhibitor DuP-769 was included as a positive control. DMSO was included as the vehicle control for determining 100% enzyme activity. Information on ELISA plate setup for anti-inflammation assays can be found in S5 Fig .

Ethics statements

Behavioral data used in this study were collected with the approval of the Uganda Wildlife Authority (permit #: COD/96/05) and the Uganda National Council for Science and Technology (permit #: NS257ES). Exportation of samples for pharmacological testing were conducted under UNCST permit #: NS104ES. Behavioral data collection adhered to International Primatological Society’s Code of Best Practice for Field Primatology [ 75 ]. No exported samples were listed under CITES. Plant samples were exported in collaboration with Makerere University (permit #: UQIS00005033/93/PC), issued by the Ugandan government, and transported to Neubrandenburg University of Applied Sciences in accordance with the Nagoya Protocol. A CUREC was approved by the University of Oxford (Ref No.: SAME_C1A_22_080). The authors report no conflict of interest.

Behavioral observations

Several unusual feeding events and putative self-medicative behaviors were recorded over 116 total field days. Table 1 reports all species collected for pharmacological testing and provides behavioral justifications for collection. Images from some of these events can be found in S1 Fig .

• PPT PowerPoint slide
• PNG larger image
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https://doi.org/10.1371/journal.pone.0305219.t001

Individuals with injuries were directly observed ingesting K . anthotheca bark and resin, W . elongata young leaves, C . alexandri bark, and C . parasitica ferns. Individuals exhibiting respiratory symptoms were observed ingesting C . alexandri bark and K . anthotheca bark and resin. Individuals with abnormal urinalysis results (e.g., positive for leukocytes, elevated ketones, and presence of blood) were observed feeding on C . patens dead wood, K . anthotheca bark and resin, and M . leucantha pith. Individuals with recent cases of diarrhea were observed consuming A . boonei and C . patens dead wood, K . anthotheca bark and resin, and W . elongata leaves. Parasitological analyses further suggest individuals with varying degrees of endoparasite infections consumed S . myrtina and C . alexanderi bark, A . boonei and C . patens dead wood, K . anthotheca bark and resin, W . elongata leaves, as well as A . polystachyus and M . leucantha pith. On a day when two individuals were observed leaf swallowing, a scientifically established self-medicative behavior, one was observed consuming K . anthotheca bark and resin, while the other was observed stripping A . polystachyus pith prior to the event. Ingestion of F . variifolia , D . dewevrei , and S . guineense bark were never directly observed during the study period. Examples of bark feeding, dead wood eating, and pith-stripping marks are shown in Fig 1 .

[ a ]: Evidence of F. exasperata bark feeding [ b ] Evidence of C. patens dead wood eating [ c ] Evidence M. leucantha pith-stripping and wadging.

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Ethnobotanical review

Based on our analysis of ethnomedicinal literature spanning various African regions from 1976 to 2022, 11 out of the 13 species tested also had documented ethnomedicinal uses ( Table 2 ).

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Production of extracts and sample information

Taxonomic information and extraction details for the 13 plant species studied, including the plant family, local name (when available), plant part used, solvent for extraction, yield of extraction, extract identification numbers (extract IDs), herbarium accession numbers, and collection location are summarized in Table 3 . Overall, the highest extraction yields were obtained with methanol-water (9/1) as a solvent. The yields from methanol-water extractions for C . parasitica , F . exasperata leaves, and S . guineense stem bark were higher than the other extractions from these samples. The plant samples which had higher yield values with n -hexane, such as the leaves of W . elongata and bark extract of A . boonei , likely have a higher content of lipids (i.e., fatty molecules).

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Library screening against multidrug-resistant human and food bacterial pathogens

Initial screening of extracts involved checking for growth inhibition against each bacterium at a concentration of 256 μg/mL. In total, 45 of the 51 plant extracts (88%) showed activity ≥40% inhibition against at least one of the 11 strains and were thus considered active and brought to dose-response experiments to determine their IC 50 value and MIC. Results from the library screening are reported in S1 Table in S2 File . As all tested plant species in the library screen had at least one extract that was active ( in vitro ) against at least one bacterial strain, no entire species was eliminated for further experimentation. However, as no extracts (at any concentration) inhibited the growth of K . pneumoniae , no further tests were conducted on this bacterium. The extract active against the most bacterial strains (n = 11) was the methanol-water extract of S . guineense stem bark (mwE098a, active against eight strains), followed by the methanol-water S . guineense leaves (mwE098b), the ethyl acetate P . patens dead wood, and the n -hexane A . boonei dead wood (hE092b) extracts, which were each active against seven, seven, and six strains, respectively. The only extract that demonstrated significant inhibition against P . aeruginosa at the highest test concentration was the methanol-water extract from S . guineense bark (mwE098a). This was also the only extract to display significant inhibition at 256 μg/mL against E . cloacae . Of all bacteria in this study, the two strains of E . coli (DSM 498 and DSM 15076) were the most susceptible, with at least one extract from all plant species inhibiting their growth. The E . coli strain with nine known antibiotic resistances (DSM 15076) surprisingly showed growth inhibition in 80% of tested extracts.

Dose-response antibacterial experiments

In dose-response assays, 41 out of the 45 tested extracts (91%) showed activity at ≤256μg/mL, though not all extracts reached MIC values (see Table 4 ). The results, along with standard deviations, are reported in S2 Table in S2 File , while S3 Table in S2 File provides a summary of the number of strains each extract was active against. The strongest in vitro growth inhibition was reported for the methanol-water extract of K . anthotheca bark and resin (mwE088) against Gram-positive E . faecium and the n- hexane extract of A . boonei dead wood (hE092b) against Gram-positive S . aureus (DSM 1104). Both extracts had low IC 50 values of 16 μg/mL (showing strong inhibition), with MIC values of 32 μg/mL against respective strains. E . faecium showed the most general susceptibility to K . anthotheca , with all extracts of this species achieving MIC values (mwE088: 32 μg/mL, eE088: 64 μg/mL, hE088: 128 μg/mL). The ethyl acetate extract of A . boonei dead wood (eE092b) also strongly inhibited the growth of E . faecium (IC 50 : 16 μg/mL; MIC: 64 μg/mL), as did the n- hexane extract of A . boonei dead wood, producing an IC 50 value of 16 μg/mL but failing to reach a MIC value. S . aureus (DSM 1104) was also highly susceptible to the ethyl acetate extracts of A . boonei dead wood (IC 50 : 32 μg/mL; MIC: 128 μg/mL).

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Only one extract, the methanol-water extract of S . guineense bark (mwE098a), was active against the gram-negative P . aeruginosa . This extract exhibited moderate growth inhibition (IC 50 : 64 μg/mL) with no MIC value reached. Despite E . coli (DSM 498) being highly susceptible on the library screen, only two extracts, the methanol-water extract of A . boonei dead wood (mwE092b; IC 50 : 256 μg/mL) and the methanol-water extract of S . guineense leaves (mwE098b; IC 50 : 128 μg/mL), reached IC 50 values at the concentration range tested, with no MICs reached. Interestingly, the strain of E . coli with nine known resistances (DSM 1576) was more susceptible, with 89% (N = 40) of extracts achieving IC 50 values ≤ 256 μg/mL. The most active extract against this strain was the methanol-water extract of K . anthotheca (mwE088; IC 50 : 16 μg/mL; MIC: 256 μg/mL). S . guineense exhibited the highest overall inhibition of S . maltophilia , with all extracts except hE098a displaying IC 50 values of ≤ 256 μg/mL against the bacterium. At the concentration range tested, no extracts yielded MIC values for S . aureus (DSM 18827), A . baumannii , E . cloacae , P . aeruginosa or E . coli (DSM 498).

Anti-inflammatory COX-2 inhibition library screen

Results from the in vitro COX-2 inhibition library screen at descending concentrations are reported in S4 Table in S2 File . At the initial concentration of 50 μg/mL, 43 out of 51 extracts (84%) exhibited an enzyme inhibition of at least 50%, displaying anti-inflammatory activity. This included at least one extract of every plant species. In the next stage of screening, at 10 μg/mL, 18 samples were eliminated. During the final step, at 5 μg/mL, five more were eliminated. The remaining 17 extracts from 10 plant species which displayed inhibition ≥50% at 5 μg/mL, were then introduced to dose-response experiments. The ethyl acetate S . myrtina bark extract (eE089b) was taken to the COX-2 dose-response despite not showing inhibition past 50 μg/mL, as it almost reached the selection limit during analysis and had a relatively high standard deviation. No extracts from W . elongata , C . patens or D . dewevrei showed COX-2 inhibition at 5 μg/mL and thus were excluded from further testing.

COX-2 inhibition dose-response experiments

The most active COX-2 inhibitors were extracts from K . anthotheca (mwE088; hE088; eE088), C . parasitica (mwE087; hE087), F . exasperata (hE093a; eE093a), S . myrtina (hE089a; eE089b), F . variifolia (eE097; hE097), A . polystachyus (hE099; eE099), M . leucantha (hE094), S . guineense (hE098a), A . boonei (hE092b), and C . alexandri (hE096). Results are reported in Table 5 . The strongest COX-2 inhibitor was the K . anthotheca methanol-water bark and resin extract (mwE088) (IC 50 of 0.55 μg/mL), followed by the C . parasitica methanol-water fern extract (mwE087) (IC 50 of 0.81 μg/mL). In contrast, all extracts of the species W . elongata , C . patens , and D . dewevrei failed to show ≥50% inhibition, mostly at the second screening concentration (10 μg/mL). W . elongata extracts notably showed low activity in both antibacterial and COX-2 inhibition assays.

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Plant species with strong pharmacological activity

This study provides the first pharmacological and behavioral evidence of its kind, based on in situ sampling, for the medicinal benefits of bark feeding, dead wood eating, and non-bitter pith stripping behaviors in Budongo chimpanzees. In the following sub-sections, we describe and discuss specific results from five of the tested plant species in further detail. For scope, we selected the two species with the strongest antibacterial properties ( K . anthotheca and A . boonei ) to profile, both of which were the only species to reach 40% inhibition at 16 μg/mL. We also selected C . parasitica to discuss as this species, along with K . anthotheca , exhibited the strongest anti-inflammatory properties. We then discuss results from our S . guineense samples, as this species was effective against the most bacterial strains in our antibacterial assays. Lastly, we selected S . myrtina , as we have behavioral evidence and health data that anecdotally support the use of this species for therapeutic self-medication by Budongo chimpanzees.

Alstonia boonei . Numerous in vitro and in vivo studies, reviewed by Adotey [ 76 ], have reported pharmacological activity in A . boonei bark. However, none of these studies investigated dead wood samples of A . boonei . Consistent with these findings, we found high levels of antibacterial and anti-inflammatory activity in the extracts of this species. Interestingly, extracts from A . boonei dead wood generally exhibited higher activity than living bark. This difference could be due either to a change in active ingredient composition, or possible fungal growth following the tree’s death. While the A . boonei dead wood n -hexane extract (hE092b) exhibited strong growth inhibition against S . aureus (DSM 1104; DSM 18827) and E . faecium at low concentrations in the dose-response assays, the n -hexane bark extract (hE092a) showed no activity <256 μg/mL. Similarly, the ethyl acetate extract of dead wood (eE092b) also strongly inhibited S . aureus (DSM 1104) (IC 50 : 16 μg/mL; MIC: 128 μg/mL) and E . faecium (IC 50 : 16 μg/mL; MIC: 64 μg/mL), while the ethyl acetate bark extract of this species did not even exhibit enough inhibition in the antibacterial library screen to be taken to dose-response assays. However, the methanol-water extract of A . boonei bark (mwE092a) did show activity against E . coli (DSM 498) (IC 50 : 128 μg/mL), as did the methanol-water dead wood extract (mwE092a) (IC 50 : 128 μg/mL), with no MIC values reached in either case. Overall, extracts from A . boonei displayed more potent activity in Gram-positive bacteria, although this effect is more apparent in dead wood than stem bark. In the COX-2 inhibition assays, the n -hexane extract of A . boonei dead wood also showed strong anti-inflammatory inhibition, while the n -hexane extract of the bark only exhibited weak inhibition (at the highest test concentration of 50 μg/mL).

A . boonei is a known medicinal plant across East Africa, commonly used for a variety of reproductive, bacterial, and gastro-intestinal issues, as well as for snake bites, asthma, and dizziness [ 68 , 76 , 77 ]. The bark and latex are intensely bitter, a reliable signal of the presence of bioactive secondary compounds and toxicity [ 94 – 96 ]. Budongo chimpanzees in both communities have been reported to consume both bark and dead wood of A . boonei , often travelling long distances to access these trees and only consuming small amounts of bark per feeding bout [ 45 ]. In an observation reported in this study (see Table 1 : A . boonei , Case 1 ), three males ingested A . boonei dead wood while outside the community’s core area for 1-minute. Two days before the event, one of the individuals had been observed with diarrhea, while also shedding visible tapeworm proglottids ( Bertiella sp.). This sample also contained unidentified protozoa, and Taenia sp. eggs. Pebsworth et al. [ 34 ] also reported an event in which four adult males, all with diverse parasite loads, traveled to a large A . boonei tree and ingested bark.

In the long-term site data, A . boonei bark ingestion was only documented 17 times between 2008–2021 [ 45 ], although this behavior was not systematically reported. In addition, the direct observation of only one A . boonei dead wood eating event, and no A . boonei bark ingesting events over the two four-month periods of observation in this study, suggest that consumption of this species is relatively rare across both communities. While specific pathogenic catalysts for selection of this species remain unknown, based on pharmacological, ethnobotanical, and behavioral data, we propose that A . boonei may be a therapeutic self-medicative resource for Budongo chimpanzees. The relatively strong inhibitory activity of this species against S . aureus , a bacteria associated with causing contamination on the skin leading to chronic wounds [ 97 ], as well as its anti-inflammatory properties, suggests that A . boonei ingestion may have beneficial effects in wound care contexts.

Khaya anthotheca . Previous studies have demonstrated that K . anthotheca bark contains biologically active compounds like gedunins, mexicanolide, phragmalin, and andirobins [ 98 ]. One limonoid identified in the species, anthothecol, has anti-cancer properties [ 99 ]. A study by Obbo et al. [ 100 ] on K . anthotheca bark collected in the Budongo Forest, found strong antiprotozoal activity against Plasmodium falciparum (IC 50 0.96 μg/mL) and Trypanosoma brucei rhodesiense (IC 50 5.72 μg/mL). A related species, K . senegalensis , has been shown to cause cell lysis in some gram-negative bacteria, including Salmonella Typhimurium , Escherichia coli , Shigella sp. and Salmonella sp., by targeting cytoplasmic membranes [ 101 ].

In our antibacterial library screen, of all extracts tested, only the methanol-water extract inhibited growth of A . baumannii (although no IC 50 values were reached in dose-response). The methanol-water extract also inhibited the growth of E . coli (DSM 498) in the library screen, as did the ethyl acetate (eE088) extract, though again no IC 50 values were reached. In our antibacterial dose-response assays, all extracts of K . anthotheca stem bark and resin exhibited strong inhibition against the Gram-positive E . faecium . The most active extract against this strain, which was also the strongest antibacterial result reported in this study, was methanol-water (mwE088) (IC 50 : 16 μg/mL; MIC: 32 μg/mL). All extracts of this species were also found to inhibit E . coli (DSM 1576) in the dose-response experiments, with the methanol-water extract once again also showing the strongest inhibition (IC 50 : 16 μg/mL; MIC: 256 μg/mL). This extract also inhibited the growth of S . maltophilia (IC 50 : 64 μg/mL) in the library screen. Only weak inhibition was found against the food pathogen S . enterica ( n -hexane extract, IC 50 : 256 μg/mL).

K . anthotheca exhibited potent anti-inflammatory activity. Of all extracts tested, the methanol-water K . anthotheca extract (mwE088) displayed the strongest COX-2 inhibition activity (IC 50 : 0.55 μg/mL). Past phytochemical studies on methanol and ethanol-water stem bark extracts from the related species, K . senegalensis , revealed many phenolic compounds, including flavonoids and tannins e.g., [ 101 , 102 ]. Flavonoids act on the inflammatory response, and may block molecules like COXs, cytokines, nuclear factor-кB and matrix metalloproteinases [ 103 ]. Some tannins have also been proven to have strong free radical-scavenging and antioxidant activities [ 104 ]. These compounds are antagonists of particular hormone receptors or inhibitors of particular enzymes such as COX enzymes [ 103 ]. If Khaya species are phytochemically similar, this could help explain K . anthotheca ’s strong COX-2 inhibitory activity.

Across Africa, K . anthotheca is traditionally used for ailments including allergies, fever, headaches, jaundice, bacterial infections, and as a disinfectant for bleeding wounds [ 105 – 107 ]. Our behavioral observations suggest that this species is also a common resource for Sonso chimpanzees, with a total of 65 feeding events recorded throughout the first field season. Of these events, several involved individuals with imbalanced health states (see Table 1 : K . anthotheca ) . On at least three independent occasions, K . anthotheca bark and resin were consumed by wounded individuals. Two adult females on different days tested positive for leukocytes on urinalysis tests within hours of ingesting K . anthotheca , suggesting the presence of infection. One of these individuals was also experiencing severe diarrhea the day prior, the other was found to have trace levels of blood in her urine. A juvenile female with a persistent cough was also observed consuming K . anthotheca bark. On several occasions individuals with high parasite loads or diverse species infection were observed targeting this resource while shedding tapeworm proglottids ( Bertiella sp.). An elderly female was also observed eating bark and resin a few hours prior to leaf-swallowing, a well-established self-medicative behavior known to rid the gut of endoparasites [ 9 , 23 ]. The frequency of K . anthotheca ingestion in the Sonso diet during this period, suggests that individuals have consistent exposure to the antibacterial and anti-inflammatory compounds present in this species. Whether this is a case of passive prevention through intake of a medicinal food, or therapeutic self-medication for a common and wide-spread condition will need further investigation. If used therapeutically, our results suggest this species could be used for treating wounds, bacterial or infections, and/or reducing internal parasite loads.

Christella parasitica.

Extracts of C . parasitica produced notably high anti-inflammatory activity in COX-2 testing, with the methanol-water extract (mwE087) achieving an IC 50 value of 0.81 μg/mL. This same extract, however, exhibited the lowest general activity in the antibacterial library screen. The only antibacterial activity from this species was on E . coli (DSM 498) by the ethyl acetate and n- hexane extracts (eE087; hE087), and on E . coli (DSM 1576) by the n-hexane extract (hE087). The n -hexane extract reached an IC 50 of 128 μg/mL in dose-response assays with no MIC value. Prior to this study, there had been limited pharmacological testing on C . parasitica (though see [ 108 ]), so comparison across studies is not possible.

When we considered the associated behavioral observation involving C . parasitica , we found a notable relevance to our pharmacological results (see Table 1 : C . parasitica , Case 1 ). This observation involved a wounded Sonso adult male (PS) travelling outside of his core area with a large group. It was unclear if this was an inter-community patrol. PS had been observed earlier in the day with a severe hand injury which impacted his mobility, though no open wound was observed. PS separated himself from the group and moved a few meters to a patch of ferns where he began consuming the leaflets. The bout lasted approximately 3-minutes. No other group members were observed feeding on this species, and this was only the second case of fern ingestion reported in Budongo in over 30-years of observations (unpublished site data). Health states of individuals from the past event were unfortunately not recorded. Whether or not C . parasitica ’s highly anti-inflammatory properties were the principal motivator for the selection of this species remains unknown, however, regardless of intention, this plant may have benefitted PS by reducing pain and swelling in his injured hand.

Syzygium guineense.

S . guineense bark and leaves have both previously been found to exhibit a range of pharmacological activity, reviewed by Uddin et al. [ 109 ]. The antioxidant, analgesic, and anti-inflammatory activities of this plant have been attributed to flavonoids, tannins, saponins, carbohydrates, alkaloids, and cardiac glycosides in the extracts [ 109 – 112 ]. In our assays, S . guineense bark exhibited high antibacterial growth inhibition effects in vitro . The methanol-water bark extract (mwE098a) showed some level of inhibition against all bacteria tested in the dose-response assays, except for E . faecium and S . enterica . This was also the only extract, out of all tested, to inhibit growth of P . aeruginosa (IC 50 : 64 μg/mL; MIC: >256 μg/mL) a pathogen known to cause infections in the blood, lungs, and other body parts after surgeries [ 113 ], and was one of two extracts to reach a MIC value against S . maltophilia (IC 50 : 32μg/mL; MIC: 256 μg/mL). The other extract to reach a MIC value was the ethyl acetate S . guineense bark extract (eE098a; IC 50 : 64 μg/mL; MIC: 256 μg/mL). All bark and leaf extracts showed strong inhibition against E . coli (DSM 1576) in the dose-response assays, with the strongest results coming from the methanol-water extracts (mwE098a and mwE098b). All bark and leaf extracts of this species, except for the n -hexane bark extract (hE098a), inhibited E . cloacae , and were the only extracts in the study to do so. E . cloacae , while part of normal intestinal flora, can cause UTI’s and respiratory infections in humans [ 114 ]. S . guineense extracts were also the only extracts to inhibit A . baumannii at a concentration <256 μg/mL, with the methanol-water bark extract showing the strongest inhibition. A . baumannii can cause infections in wounds, blood, urinary tracts, and lungs [ 115 ]. The efficacy of methanolic extracts from this species suggests that the active compounds are polar molecules. In the anti-inflammatory COX-2 inhibition dose-response assays, only the n -hexane bark extract displayed strong inhibitory effects (IC 50 : 2.42 μg/mL), while the other extracts failed to exhibit significant activity during the pre-screening or ≥ 50% inhibition at 10 μg/mL. The COX-2 inhibition assays showed no inflammatory inhibition amongst leaf extracts at tested concentrations.

S . guineense can be found throughout Sub-Saharan Africa and is a common traditional medicine, for malaria [ 116 ]. The bark is also used for stomach aches, diarrhea, internal parasites, and infertility [ 68 , 109 ]. Ingestion of S . guineense bark is rare in Budongo, with no direct observations in either community throughout the study period, and only six total cases between 2008–2021 documented in the site’s long-term data. No observations of leaf ingestion of this species have ever been reported. The infrequent ingestion of S . guineense bark implies a more targeted use, making it unlikely to be a medicinal food. Instead, our pharmacological findings make this resource a strong candidate as a putative, therapeutic self-medicative resource. Unfortunately, as there is currently no health data associated with individuals who have recently consumed S . guineense bark, we do not yet know which properties chimpanzees may be targeting. However, based on pharmacological results, we recommend further investigation into this species as a curative agent for respiratory-related infections.

Scutia myrtina.

Kritheka et al. [ 117 ] in their study on the bioactivity of S . myrtina , found in vivo evidence that this species possesses dose-dependent anti-inflammatory, antimicrobial, and antifungal properties. Across our antibacterial assays, the bark sample of this species collected from the stem inhibited E . faecium (eE089a) and E . coli DSM 1576 (eE089a; mwE089a) in dose-response tests at concentrations ≤256 μg/mL. The refuse sample, collected from the ground below the plant’s stem, inhibited A . baumannii (hE089b), E . faecium (eE089b), and E . coli DSM 1576 (mwE089b; eE089b; hE089b) in dose-response tests below the specified concentration. Interestingly, the refuse sample inhibited more bacteria species overall than the fresh bark. The most potent antibacterial growth inhibition effects came from the ethyl acetate bark sample against E . faecium (eE089a; IC 50 : 64 μg/mL), though no MIC value was reached. In the COX-2 inhibition assays, the n- hexane bark extract had the fifth strongest inhibitory effect in vitro (hE089a; IC 50 : 1.19 μg/mL) out of all samples, while the ethyl acetate refuse bark sample was less potent, though still moderately active (E089b; IC 50 : 7.49 μg/mL).

As far as the authors know, this is the first published report presenting both behavioral and pharmacological evidence for S . myrtina bark as a putative medicinal resource amongst free-ranging chimpanzees (though see [ 118 ] for evidence based on food-combinations). Our behavioral observations indicate that an individual with a diverse and intense parasite infection deliberately sought out the bark of this species. The Budongo chimpanzees may, therefore, utilize S . myrtina as an anthelminthic. Across traditional accounts from multiple regions, S . myrtina is commonly used by people as an anthelminthic to treat intestinal worms [ 68 ], while aerial parts are also used to treat various bacterial infections. As we were not able to conduct urinalysis on the consumer during or after this event, we cannot determine whether the individual also harbored a bacterial infection at the time of ingestion. However, this possibility cannot be ruled out. Based on these findings, we propose S . myrtina be added to the list of putative chimpanzee self-medication behaviors as a treatment for internal parasites, and we encourage further exploration into the other specific chimpanzee health conditions that this species may help ameliorate.

Assessment of putative self-medicative behaviors

We synthesized pharmacological and behavioral evidence to assess therapeutic use of species associated with bark feeding, dead wood eating, and pith stripping behaviors. A summary of the antibacterial and anti-inflammatory results for each species is reported in S3 Table in S2 File . Overall, stem bark and dead wood samples were notable for their activity. Bark samples from every species showed >40% antibacterial inhibition against at least one bacterial strain. This activity was also true of the dead wood samples. When plant parts of the same species were tested ( S . guineense and F . exasperata ), barks generally exhibited more potent antibacterial and COX-2 inhibition activity than the leaves, likely to do with the higher concentration of plant secondary metabolites in bark. Our findings offer strong support that bark and dead wood eating of certain species could constitute novel self-medicative behaviors in wild chimpanzees. We also encourage more investigation into the bioactivity of non-bitter pith stripping, as the pith of A . polystachius showed strong antibacterial activity against E . faecium (hE099; IC 50 : 32 μg/mL; MIC: 128 μg/mL), and the piths of both A . polystachius and M . leucantha demonstrated significant anti-inflammatory properties at low concentrations. Future primatological research should prioritize the establishment of multi-disciplinary long-term projects that look systematically at health states of individuals who engage in bark, dead wood, and pith ingestion behaviors. We also encourage further pharmacological testing on other species used for these behaviors in Budongo and across primate field sites.

Drug discovery

Multidisciplinary studies on this topic have potential to lead to the discovery of new medicines which may benefit our own species [ 119 – 122 ]. Historically, PSMs have played a major role in the development of modern human medicine, and even today, a large portion of medicines are derived either directly or indirectly from plants and other natural materials [ 123 – 127 ]. Antimicrobial resistance is rising to dangerously high levels according to the World Health Organization [ 128 ] requiring the rapid creation of new antibacterial treatments. Infections caused by multi-drug resistant bacteria kill hundreds of thousands of people annually. Our findings of strong antibacterial growth inhibition across numerous plant species growing in Budongo have promising implications for our ability to discover novel compounds in existing forest habitats. Extracts should also be tested against additional bacteria and for anti-virulence effects, e.g., inhibition and disruption of biofilm formation, quorum sensing and toxin production, pursuing development of new therapeutic strategies that apply less evolutionary pressure, likely resulting in emergence of less antibiotic resistances in the future. Phytochemical characterization using advanced techniques, such as LC-ToF-MS and NMR, as well as potentially AI-assisted untargeted metabolomics approaches, are now needed to identify substances present in the most active extracts. This may eventually lead to the isolation and structure elucidation of yet unknown active ingredients and make way for determining their pharmacological selectivity and toxicity, while also taking potential synergistic effects into account.

Simultaneously, we are currently faced with a pressing need for more effective treatments to combat symptoms of acute inflammation and mediate long-term consequences of chronic inflammatory diseases [ 129 ]. The prostaglandin-producing cyclooxygenase-2 (COX-2) mediates and regulates pain, fever, wound inflammation, and many other medical disorders, as it plays a crucial role in the host organism’s defense against pathogens and injury. COX-2 inhibition has the same mechanism of action as non-steroidal anti-inflammatory drugs (NSAIDs). While inflammation is a normal part of the body’s defense against injury or infection, it can be damaging when occurring in healthy tissues or over a protracted period. Chronic inflammation can lead to cardiovascular diseases (CVD) and cancer, the two leading global causes of death [ 130 ]. Past studies have shown that the IC 50 values of Aspirin and ibuprofen (pure compounds and common NSAIDs) are 210 μg/mL and 46 μg/mL respectively for COX-2, and 5 μg/mL and 1 μg/mL respectively for COX-1 [ 131 , 132 ]. The in vitro COX-2/COX-1 selectivity ratio for Aspirin and ibuprofen is 42 and 46 respectively. Surprisingly, the 17 most active extracts in our COX-2 assays display lower IC 50 values than these popular NSAIDs, meaning our extracts have more potent inhibitory effects on the inhibition of COX-2 than the most common anti-fever and anti-pain drugs on the market. While COX-1 assays were beyond the scope of this study, future research should investigate COX-1 inhibition activity of these 17 extracts to calculate COX-2/COX-1 selectivity ratios. Doing so will allow for preliminary assessment of potential side effects, selectivity, and efficacy before future in vivo experiments can commence.

Future directions

Future research on this topic would benefit from the inclusion of control samples (plants or plant parts not consumed by chimpanzees); however, in this study, assay costs were a prohibiting factor. Additional information regarding the nutritional and mineral content of the species mentioned in this study is needed to better understand the motivations for ingestion. However, bioactivity and nutritional/mineral content are by no means mutually exclusive. It is, therefore, highly likely that these resources provide multiple benefits to consumers.

Future studies should also consider ecological variables. For example, different individual plants of the same species should be tested across habitat types to determine whether bioactivity varies based on location, age, life history, or time of harvest. Situating samples in their ecological context will provide a better understanding of whether chimpanzees select resources based on species alone, or other more nuanced criteria. Lastly, climatic studies in combination with pharmacological testing should examine how climate change may impact bioactivity of these plants, as shifting weather patterns have already been shown to alter nutritional content [ 133 ]. This information will be critical for establishing protected habitats that can sustain healthy, wild, primate populations.

Conclusions

As we learn more about the pharmacological properties of plants ingested by chimpanzees in the wild, we can expand our understanding of their health maintenance strategies. Our results provide pharmacological evidence, from in vitro assays of plant parts consumed by wild chimpanzees collected in situ , for the presence of potent bioactive secondary plant metabolites in Budongo chimpanzee diets for a variety of potential illnesses previously not considered. Whether these resources are consumed intentionally as a form of therapeutic self-medication or passively as medicinal foods, must be assessed on a case-by-case basis, taking behavioral observations into account.

For the field of zoopharmacognosy to progress, we encourage continued multidisciplinary collaboration between primatologists, ethnopharmacologists, parasitologists, ecologists, and botanists [ 9 ]. Beyond improving our broad understanding of chimpanzee health maintenance, multidisciplinary studies will benefit our own species, potentially leading to the discovery of novel human medicines to combat the looming problem of growing drug-resistance. For this to happen, however, it is imperative that we urgently prioritize the preservation of our wild forest pharmacies as well as our primate cousins who inhabit them.

Materials availability

Voucher specimens for each species were deposited at the Makerere University Herbarium in Kampala, Uganda for taxonomic identification and storage. A duplicate set was deposited at the University of Oxford Herbarium for permanent storage.

Supporting information

S1 fig. budongo chimpanzees consuming resources tested in this study..

a.) IN eating K . anthotheca bark and resin b.) MZ eating S . myrtina bark c.) KC stripping A . polystachyus pith d.) MB eating C . patens dead wood e.) OZ eating S . guineense bark (post-study period) g.) MZ eating F . exasperata bark.

https://doi.org/10.1371/journal.pone.0305219.s001

S2 Fig. Generalized multi-method workflow used in this study.

https://doi.org/10.1371/journal.pone.0305219.s002

S3 Fig. Voucher samples collected in duplicate.

a . ) C . alexandri (00243133G) b . ) A . polystachius (00243136J) c . ) W . elongata (00243129L) d . ) C . parasitica (00243122E) e . ) K . anthotheca (00243123F) f . ) F . variifolia (51195) g . ) M . leucantha (51203) h . ) A . boonei (51204) i . ) D . dewevrei (00243132F) j . ) S . guineense (00243135I) k . ) S . myrtina (00243128K) l . ) F . exasperata (00243130D).

https://doi.org/10.1371/journal.pone.0305219.s003

S4 Fig. Plate layouts for growth inhibition assays.

[Top] Library Screen: done in 96-wells-mikrotiterplate; AB: Antibiotic as positive control; DMSO: vehicle control / negative control; GC: growth control: containing working culture, to check whether the bacterium grew/active; [Bottom] Dose-Response: done in descending concentration of samples, DMSO, and antibiotic. MB: Media blank, consisted of CAMHB as negative/ sterile media control; DMSO as negative/ vehicle control; GC: growth control, consisted of working culture.

https://doi.org/10.1371/journal.pone.0305219.s004

S5 Fig. ELISA assay setup for anti-inflammatory assay.

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S1 File. Supplementary materials: Methods .

https://doi.org/10.1371/journal.pone.0305219.s006

S2 File. Supplementary tables.

https://doi.org/10.1371/journal.pone.0305219.s007

Acknowledgments

We are grateful to all the field staff working in Budongo who provided invaluable instruction and guidance, generously sharing both scientific insight and traditional knowledge. This study could not have been done without their contributions. Specifically, we would like to thank members of the Perspectives Collective: Chandia Bosco, Monday Mbotella Gideon, Adue Sam, Asua Jackson, Steven Mugisha, Atayo Gideon, and Kizza Vincent, and Walter Akankwasa, as well as site director David Eryenyu. We would also like to thank Godwin Anywar for his assistance with plant identification at the Makerere Herbarium, Stephen Harris at the University of Oxford’s Herbarium for his facilitation of voucher storage, and the Natural History Museum in London for their aid in parasite identification. We are grateful to Vernon Reynolds who founded the field site and to the Royal Zoological Society of Scotland for providing core support. We also gratefully acknowledge the Uganda Wildlife Authority and the Uganda National Council for Science and Technology for granting permission to conduct research in Uganda. Lastly, thank you to the staff and students at Neubrandenburg University of Applied Sciences who made this collaboration possible, and to research assistant, Finn Freymann, for his help with botanical extractions.

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