Culturable endophytic fungal communities associated with plants in organic and conventional farming systems and their effects on plant growth

As compared to organic farming system, conventional farming system relies on higher inputs of synthetic agrochemicals, which may reduce the abundance, diversity, and beneficial effects of plant endophytic fungal communities. This study compares the diversity and abundance of culturable endophytic fungal communities associated with four plant species –corn, tomato, pepper, and watermelon grown in separate organic and conventional fields. In all, 740 fungal isolates were identified, of which 550 were from the organic fields and 190 from the conventional ones. These fungal isolates were grouped into eight orders and 22 species, with the two most abundant species being Trichoderma sp. and Pichia guilliermondi. The fungal species diversity and abundance were both significantly higher in the organic than in the conventional fields. All the isolated endophytic fungi improved tomato plants’ shoot growth and biomass significantly, as compared with the water control. Six fungal isolates also exhibited activity that enhanced tomato fruit yields. These results suggest that these endophytic fungi might be a considerable boost to sustainable agricultural production, while also reducing the agricultural application of chemicals and thus benefiting the environment and human health.


Results
Community composition and abundance. A total of 740 culturable endophytic fungal isolates were obtained from tomato, corn, watermelon, and pepper plants collected from the organic and conventional fields. Of these, 28 different fungal isolates were identified based on the top hits for Internal Transcribed Spacer (ITS) sequences in the National Center for Biotechnology Information (NCBI) database, and morphology phenotypes grown on potato dextrose agar (PDA) plates 4 . The whole samples of 740 fungal isolates were subdivided into 22 different species, which were further classified into eight distinct orders (Fig. 1A,B). The supplementary figure (Fig. S1) shows the phylogenetic tree with the accession numbers of the matched rDNA-ITS sequences of these 28 fungal isolates from the NCBI in parentheses, and the BLASTn expected values and percent-sequence identities in brackets by the method reported previously 29,30 . The results confirm that these isolates are separate species, a conclusion supported by bootstrap values. For example, the Pleosporales sp. (GQ923961) and Pleosporales sp. (GQ924020) were clustered together with a bootstrap value of 92%. Similarly, Aspergillus nidulans (HQ285615) and Aspergillus nidulans (EU409807) were in the same group, with a bootstrap value of 97% (Fig. S1).
At the order level, the Hypocreales were the most common, which included 352 isolates and seven species, around 48% and 32% of the total fungal isolates and species, respectively (Fig. 1A,B). The Eurotiales and Saccharomycetales were the second and third most abundant orders, and together constituted approximately 20% Scientific RepoRts | (2019) 9:1669 | https://doi.org/10.1038/s41598-018-38230-x and 14% of all isolates and species, respectively (Fig. 1A,B). The other identified orders were the Pleosporales, Trichosphaeriales, Chaetothyriales, Sordariales, and Xylariales (Fig. 1A,B). Interestingly, the most common fungal species across all crops and field types was Pichia guilliermondi, followed by Thrichoderma spirale and Thrichoderma atroviride. However, the number and distribution of fungal species varied considerably by plant species (Fig. 1C-F). The abundance and species diversity of culturable endophytic fungi were both significantly higher in the organic fields than in the conventional ones. Of the 740 total isolates, 550 (74.3% of total) were obtained from plants grown in the organic fields ( Fig. 1C-F). A total of 18 fungal species were distributed in both field types, but four species-Chaetomium globosum, Fusarium sp., Periconia macrospinosa, and Rhinocladiella sp. were only found in the organic ones ( Fig. 1C-F). Similarly, of the eight orders, six were found in both fields, but the Chaetothyriales and Sordariales were unique to the organic fields (Fig. 2). The species diversity of the endophytic fungi associated with corn, pepper, and melon plants in the organic fields was significantly higher than the same plant species in the conventional fields. In the case of tomato plants, such species diversity was also higher in the organic fields, but this difference was not significant ( Table 1). The abundance of the endophytic fungal species associated with all four plant species in this study was significantly higher in the organic fields than in the conventional ones (Table 2).
Fungal species diversity and abundance associated with specific tissues. Eight fungal species were only found in plant root and shoot tissues, but not seed tissues, while the other 14 were identified in all three major plant-tissue types (Fig. 3). As measured by the Shannon-Weiner Diversity Index, root tissues in this study displayed the highest level of fungal species diversity and seed tissues had the lowest level (Table 1). More specifically, the root community included 48% of the isolates (n = 365), including all 22 species; the shoot community, 38% of the isolates (n = 275), again including all species; and the seed community, 14% of the isolates (n = 100), which belonged to less than two-thirds of the species (n = 14) (Fig. 3). Analysis of variance (ANOVA) revealed  that differences in the abundance of endophytic fungi across shoot, root, and seed tissues in both field types were significant (Table 3).
Fungal isolates showed the promotion activity for plant growth and fruit yields. As noted above, one of the main aims of this study was to identify endophytic fungi that could be cultured and applied to plants to enhance their growth, health, and yields. Therefore, we tested 28 different fungal isolates for their ability to enhance tomato plants' shoot growth and biomass. All 28 isolates were found to do so: with the plants' heights, fresh weights, and dry weights all significantly greater than those of the plants treated with water control (Tables S1-S3). Detailed information about the ITS sequences and NCBI accession numbers of these fungi is provided in Figure S1 and Table S4. Among the top 10 fungi in terms of their promotion of tomato shoot height, six also were found to improve tomato fruit yield. These were Coniothyrium aleuritis isolate 42, Pichia guilliermondii isolate F15, Fusarium oxysporum strain NSF2, Fusarium proliferatum strain AF04, Aspergillus nidulans strain FH5, and Trichoderma spirale strain YIMPH30310 (Fig. 4).

Discussion
Organic farming practices have developed rapidly worldwide over the past 20 years because of their lower inputs of chemicals and their benefits to crop health and the environment 14,31 . In particular, these practices directly affect soil microbial abundance, diversity, and functions, and thus are likely to be associated with the increased soil health, enhanced plant growth and yields, and improved plant resistance to biotic and abiotic stresses 13 . The present study revealed that the abundance of endophytic fungal species in the sampled organic fields was significantly higher than that in the sampled conventional ones for all four studied plant species. It was also observed that the plant-associated endophytic fungal community had significantly higher species diversity in the organic fields. The data also suggested that these two agricultural systems differentially influenced the abundance and diversity of the plant-associated endophytic fungal community, and potentially its impacts on plant growth and yields. This echoes previous studies' findings that fungal endophytes of grapevines and soybeans had higher abundance and species diversity in organic fields than in the conventional fields 32,33 .
In addition, the present study's results demonstrate that endophytic fungal species' diversity and abundance differed across different plant tissues, being significantly higher in roots than in shoots or seeds. This might be because roots are the interfaces that connect plants with the soil and the soil-associated microbiome, including some soil microbes that could potentially be plant endophytes 34,35 . Similar results have been reported previously: for example, in regard to the herbaceous grassland plants, medicinal plant Kadsura angustifolia, and    [36][37][38] . Prior studies have also shown, however, that fungal endophytes can be more abundant and diverse in plant tissues other than roots 39,40 . The reason for this discrepancy might be that the distribution of fungal endophytes across different tissue types is affected by different plant species, growth stages, soil types, and environmental conditions 41 . One of our main aims for this study was to identify the beneficial plant-associated endophytic fungi to increase plant growth, heath, and yield by a sustainable approach. The two most abundant fungal species isolated in this study were Pichia guillermondii and Trichoderma sp. in total, which were isolated from both the organic and conventional fields in our study. The beneficial effects of Pichia guillermondii and Trichoderma sp. on enhancing plants' growth, yields, and resistance to biotic stress have been extensively reported by previous researchers 24,25,42,43 . For example, several P. guillermondii strains had been reported to significantly inhibit the growth of Penicillium digitatum, the pathogen that causes green-mold diseases in citrus and many other crops. Moreover, after treatment with these P. guilliermondii strains, the peroxidase activity in the flavedo tissues of citrus was significantly higher than that in the untreated controls, and the related activity was associated with the increased resistance to Penicillium digitatum. Thus, P. guilliermondii could potentially trigger plants' defense mechanisms against pathogen attacks 42 . Additionally, P. guilliermondii have been shown to promote the growth of diverse plants, such as chili peppers 24 . Trichoderma sp., meanwhile, have been widely found to promote plant growth and inhibit a range of plant pathogens, including Sclerotium rolfsii, Verticillium dahlia, and Fusarium sp. 25,[43][44][45] . In addition to promoting tomato plants' growth, T. atroviride and T. harzianum have been reported to activate the plants' defense-related hormones, such as the salicylic acid (SA) pathway even without the presence of pathogens. They could also activate the jasmonic acid (JA) signaling pathway after attack by the fungus Botrytis cinera 43 . T. spirlae fungi have been identified as phosphate solubilizers and could promote the growth of plants, including eggplants under field conditions 25 .
The six fungal isolates in current study associated with the highest increases in tomato growth and yields belonged to very diverse orders, i.e., the Eurotiales, Pleosporales, Hypocreales, and Saccharomycetales. This implies that a very diverse range of endophytic fungi have considerable potential to improve plant yields and health under natural conditions. Beneficial fungi may increase plants' growth and yield via various mechanisms: for example, through increasing their uptake and utilization of nutrients, such as nitrogen, phosphate, and iron; promoting their growth hormone production; and activating the genes involved in their growth and development 5,24 . However, the precise mechanisms whereby these six endophytic fungal isolates can improve tomato plants' growth and yields merit further investigation. It is expected that the outcomes of such future research will lead to the development of a more efficient strategy for utilizing fungal endophytes in sustainable agriculture.
As noted above, plants' endophytic fungal communities can change in response to diverse factors, including their growth stage and environmental conditions 46 . The present findings highlight that organic as opposed to conventional practices could increase the abundance and species diversity of culturable plant-associated endophytic fungi. Further study, using next-generation sequencing approaches, of the diversity and abundance of the endophytic fungal microbiome in plants grown in organic versus conventional systems, is also likely to improve our understanding of the obligate and unculturable endophytic fungi in the community. It would also be worthwhile to enhance microbial interactions at the community level by creating synthetic beneficial microbial communities from isolated culturable fungal microbes, as this might benefit plants more efficiently than single microbe would.
More comprehensive studies of the effects of various organic-farming practices, such as the use of cover crops, rotation, tillage, or crop integration, could help identify the best practices for promoting the abundance and diversity of the soil-and plant-associated microbiomes, which in turn should benefit plant growth and yields. Such further investigations would also facilitate more efficient applications of beneficial endophytic microbes in organic and sustainable agriculture practices.
Prior findings discovered that the differences in agricultural practices could strongly influence the abundance and composition of soil-associated microbiomes [47][48][49][50] . One reason for this is that the differences in water use across different farming types can affect soil environment in complex and interactive ways. For instance, the survival and growth of microbes in soil can be directly affected by the soil water content, which can also influence microbes' access to nutrients that are essential for their survival [47][48][49] . Another key factor is fertilizer application. For example, the amount and quality of organic fertilizers could both play critical roles in soil microbial diversity 50 . Microbial-community differences between organic and conventional farming systems could also be more significant when pesticide application is large, soil-tillage operations occur, and cropping systems lack soil-replenishing crops, such as legumes 50 . Since some soil microbes might potentially become plant endophytes 34,35 , these agricultural practices could also affect the plant-associated endophytic microbial community, as our present study. It is important to remember, however, that the soil-and plant-associated microbiome is not limited to fungi, but also includes bacteria and other microorganisms, which might not be culturable in certain growth media. Yet, these microbes' interactions with one another may affect not only their own survival, but the overall community's impact on plant and soil health, in ways that remain under-researched and poorly understood 34,51 .
Some prior research has suggested that tillage practices could affect the C:N ratio of the microbial biomass in soil, but only cause significant differences in fungal dominance over bacterial dominance when soil aggregations change 52,53 . One study has also shown that some plant endophytic fungi have a greater capability to solubilize inorganic phosphates, and produce more active enzymes, than endophytic bacteria do, meaning that the former could lead to higher plant biomass than the latter 54 . In addition, fungi have exhibited desiccation responses in soil, which might indicate their higher degree of resistance to water-availability changes, as compared to bacteria 55 . However, further study should explore the effects and functional mechanisms of fungi versus bacteria in different crops and agricultural systems under a variety of environmental conditions.
Taken as a whole, the present study's results indicate that organic farming practices can have significant positive effects on the abundance and diversity of plant-associated culturable endophytic fungal communities. All the endophytic fungi that were isolated and cultured during this study exhibited significant beneficial effects on tomato plants' shoot growth and biomass, and six were also found to significantly increase the plants' fruit yield following soil-drench treatment with the broth of fungal spores and mycelia. The results suggest the strong potential of these endophytic fungal microbes to benefit agriculture, not only through the promotion of crop growth and yields, but also through the reduction of chemical inputs. However, further investigation of the mechanisms by which the beneficial functions of these fungal endophytes occur, and how to utilize them efficiently in practice, will be critical to the success of their further application in sustainable agricultural production. Isolation of the endophytic fungi from the plant shoot, root, and seed tissues. The approach to isolate the endophtyic fungi from the plants was followed by the widely reported method through the plant surface sterilization 23,56-58 . The same weight of 0.5 gram of shoots (leaf/stem) and root segments with approximately 1 cm-1.5 cm in length were taken from the collected 20 plants for four different plant species in both the organic and conventional fields. These segments were washed with deionized water (dH2O) to remove the remnant soil and debris. The segments were immersed into 20% Clorox bleach (sodium hypochlorite) containing 0.1% Tween 20 (Sigma-Aldrich Co., St. Louis, Mo.) in sterile dH 2 O for 15 mins, rinsed with 95% ethanol (EtOH) for 2 mins, and then serially rinsed 5 times in sterile dH 2 O 9,56-58 . The same amounts of seeds were excised from the lemma and palea of the collected plants, cut into two parts, and then surface sterilized as above. To test the efficacy of this method, the final step dH 2 O rinsed off the plants was randomly collected and plated on the PDA (Potato Dextrose Agar) plates and incubated at 26 °C for 15 days to confirm the absence of any microbial growth after the disinfection.
All the cut segments of different tissues from each plant were placed separately on the plates with the PDA medium containing the ampicillin, penicillin, and streptomycin (30 μg/ml) to inhibit the bacterial growth (Fisher Scientific, USA). The plates were then incubated in the growth chamber with the setting temperature at 26 °C for 7-12 days and the occurrence number of individual fugal colonies emerged were recorded separately from different tissues and different plants to further calculate the fungal abundance and species diversity 23,56,59 . The single colonies were cultured separately on plates with the repeats for 2-3 times to get the pure culture for further study.
DNA extractions, ITS rDNA gene amplification, sequencing, and OTUs identification. The DNA from the purified fungal isolates grown in PDA plates was extracted by using the Zymo Research fungal/bacterial DNA mini-prep kit (Zymo Research, CA, USA) following the manufacturer's instruction. The ITS (Internal Transcribed Spacer) region is the most widely sequenced DNA region for the fungi and it has higher degree of variation than most of the other genic regions of rDNA 60,61 . Therefore, the ITS sequencing method had been selected for our study. The rDNA amplification of the ITS region was performed in a 50 µl reaction mixture, which included 3 µl DNA template (1-20 ng), 50 µM of primers ITS1 (5′-TCCGTAGGTGAACCTGCGG -3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC -3′) 29,34,60-64 , 3 mM Mgcl 2, 3 mM dNTPs, 5 µl of Taq buffer, and 1 U Taq DNA polymerase (Fermentas Inc., MD, USA). The PCR amplification was performed on a cycler PCR machine (Bio-Rad Labortories, CA, USA) with the initial denaturation at 94 °C for 5 mins, followed by 30 cycles of amplification (94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min) and an extension step (72 °C for 5 mins) 23 . The PCR products around 600 to 700 bps were purified using the Zymo PCR purification kit (Zymo Research, USA) and quantified by a nano-drop spectrophotometer. The purified products had been sent to the eRAMP genome center at The Ohio State University for sequencing by the Sanger sequencing method 65  Data organization and statistical analysis. As well as being sorted by species, the fungal isolates were grouped into a higher taxonomic level (orders); and their relative abundance was evaluated according across three major types of plant tissues and two general types of agricultural systems (conventional vs. organic). Multiple alignments of the rDNA-ITS sequences were generated using multiple-alignment fast Fourier transform 66 and the related phylogenetic tree was constructed by the maximum-likelihood method RAxM 67 . Species diversity was calculated using Shannon-Weiner Diversity Index values 68,69 and the Hutcheson t-test and ANOVA were used to compare the relative abundance of fungal species across shoot, root, and seed tissues and between the organic and conventional systems 69,70 . Dunnett's test was used to analyze the effects of different fungal isolates on tomato plants' shoot growth, weights, and fruit yields 71 .
Test of the plant growth promotion and yield enhancement activities of different endophytic fungal isolates. The tomato seeds (var. OH981205) were washed with 95% EtOH in dH 2 O for 2 mins and were soaked with 30% bleach (NaOCl) and 5% sodium dodecyl sulfate (SDS) in dH 2 O for 20 mins. The seeds were further rinsed with the sterilized dH 2 O 5 times and incubated at 4 °C in a cold room for 24 hrs. The washed tomato seeds were then sown into pots that contained the Pro-Mix potting media (Premier Horticulture Inc., PA, USA). The pots were kept in a greenhouse with the constant temperature of 24 °C with 16 hrs of light followed by 8 hrs of darkness for 7 days for the seed germination 23 . Then, the tomato seeds were inoculated with the fungal inoculation mixture containing fugal mycelia and spores (if present) in 100 ml water in each pot as described below and previously 62 . Each pot contained one tomato seed and six pots were used for each fugal treatment and water treatment as controls. The related experiments had been repeated for three times. For the fungal inoculation mixture preparation: the pure and different individual 28 fungal isolates were maintained and cultured on the petri dishes filled with PDA media. For each endophytic fungal isolate, three fugal plugs with diameter around 0.6 cm were taken from the culturing PDA plates and put in the center of three new PDA medium plates separately and grown in a 26 °C incubator for 7 days. Then the fungal spores (if present) and mycelia from three plates were washed off with the sterilized water and put into the sterilized flasks and diluted to a total of 600 ml. About 100 ml of the fungal broth containing the fungal spores (if present) and mycelia were applied to the top of the potting mix in each pot containing the tomato seed underneath; and 600 ml water treatment were used as controls in separate 6 pots with 100 ml for each pot 62 .
All the pots were kept in a greenhouse with a constant temperature of 24 °C and exposed to 16 hrs of light per 24-h period. The tomato plants' shoot growth (height) and biomass (fresh weight, and dry weight after being placed in an oven at 70 °C for 48 hrs) were measured at around 10-week growth stage. The experiments were repeated three times, and the data from 18 plants in each treatment condition were pooled together for further analysis. The comparison of tomato plants' above-ground shoot heights, dry weights, and fresh weights across each fungal treatment and water-treatment control were analyzed by using Dunnett's test 71 .
The top 10 fungal isolates in terms of their positive impact on tomato plants' shoot heights were selected for further testing of their effects on tomato fruit yields. Six plants in each of the 11 treatment conditions (i.e., for each of the top 10 fungi plus the water control) were chosen for measuring. The combined weight of several batches of tomatoes harvested from each plant during the entire growing season was counted as the total tomato fruit yields. The experiments were repeated three times and the data for 18 plants were used for further analysis.