Farmyard Mulching in Regenerative Agriculture Enhances Saprotrophs and Concomitantly Reduces Pathogenic Fungal Genera

Farmyard Mulching in Regenerative Agriculture Enhances Saprotrophs and Concomitantly Reduces Pathogenic Fungal Genera | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Farmyard Mulching in Regenerative Agriculture Enhances Saprotrophs and Concomitantly Reduces Pathogenic Fungal Genera Pratyusha Naresh , View ORCID Profile Indira Singh doi: https://doi.org/10.1101/2024.08.14.607896 Pratyusha Naresh 1 People’s Education Society (PES) University , Bangalore Find this author on Google Scholar Find this author on PubMed Search for this author on this site Indira Singh 2 Indian Institute for Human Settlements (IIHS) , Bangalore Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Indira Singh For correspondence: indira.singh{at}iihs.ac.in Abstract Full Text Info/History Metrics Preview PDF Abstract Regenerative agriculture (RA) using nature-friendly methods nurtures soil microbial communities. Indian RA farmers use diverse practices to manage their soil. This study compared the fungal communities in RA plots with those in conventional agriculture (CA) and barren land (BL) plots (comprising completely barren-BL and with Eucalyptus - BL-Euc). Two crops were considered - Finger millets and Vegetables (Tomato/ beans) for this study. ITS metagenomic analysis of soil DNA samples obtained from RA, CA and BL plots was done to identify fungal composition in each of the study plots. The fungal communities in RA finger millet and RA vegetable were compared with respective CA finger millet and CA vegetable and with BL plots. Vegetable RA plots observed higher abundances of fungal Operational Taxonomic Units (OTUs) than in CA vegetable and BL plots. Whereas the RA finger millet plots had similar fungal OTUs as in CA finger millet and BL plots. The vegetable RA plot carrying out natural farming for 12 years (maximum length in our samples) recorded the highest fungal OTU (13707) levels while the CA plots had average OTU abundance of (7416). RA plots in both crops showed a significant reduction in plant pathogenic fungal genuses - Bipolaris and Pyrenochaetopsis . Furthermore, RA finger millet plots showed an enhanced representation of saprotrophs while CA (finger millets) had pathotroph-saprotrophs suggesting a favorable increase in decomposer populations in RA. Introduction With the world moving towards sustainability in agriculture through natural/ regenerative practices, soil and plant microbial health is gaining more and more prominence. Healthy soil boasts of millions of different species of bacteria, fungi, earthworms, nematodes, insects and other invertebrates ( De Gannes et al., 2016 ; Costantini and Mocali, 2022 ; Bach et al., 2020 ). These organisms together help to build the soil’s texture, porosity, water retention capacity, and quality through nutrient cycling processes, in effect supporting plant growth and productivity ( Schröder et al., 2016 ). Fungi play significant roles in dictating soil health and agricultural outcomes ( Frąc et al., 2023 ). In an agricultural ecosystem, fungi carry out essential processes involving decomposing the organic matter and enriching the soil with minerals, making them accessible by the plant roots by solubilizing these minerals. In addition, they release several chemicals such as siderophores and indole acetic acid promote plant growth, and associate with plant roots enabling better mineral and water uptake, and finally also protect plants against pests and pathogens ( Selvasekaran and Chidambaram, 2020 ). Many fungi are directly associated with the plants as endophytes enhancing the plant’s health and productivity ( Poveda et al., 2022 ). Several others get into symbiotic relationships with plants and algae such as lichens, which help plants by supporting soil formation, nitrogen fixation, and fixing carbon dioxide through the photosynthesizing algae ( Du et al., 2019 ). However, there is also a host of pathogenic fungi that end up destroying the crops and reducing their productivity ( Doehlemann et al., 2017 ). Interestingly, nature provides us with a plethora of diverse solutions to overcome pests and pathogenic species. Among the fungal kingdom also a large number of species have been recorded with anti-pathogenic properties ( Pandit et al., 2022 ). Climate change has been overburdening the pest problem ( Hyde, 2022 ). Hence, in recent scenarios analysis of the soil fungi is essential to arrive at nature-based solutions for resolving the climate mediated agricultural challenges. Regenerative agriculture uses a wide variety of soil enriching practices that dissuade the use of chemicals but rely on organic inputs such as farmyard waste, compost, crop mulch etc. These dead organic matters form a rich resource for the growth and multiplication of saprophytic microbes. Going with this hypothesis this research assessed the fungal community structure in natural/ regenerative agriculture (RA) and compared it with the conventional agricultural plots and barren land and eucalyptus containing plots. Like the bacterial populations in RA soils which were seen to host a more diverse community of bacteria with increase in prominent sets of Plant Growth Promoting Rhizobacteria (PGPR) genera, similarly here we have explored the impacts of healthier agriculture practices on fungal profiles. Materials and Methods Soil Sampling and Chemical Analysis Two crops were considered for the study: ragi (finger millet) and vegetables (tomato/beans). This study is an extension of the previously published - Singh et. al., 2023, which analyzed the bacterial community structures in the plots. We have three types of plots - Regenerative (RA), Conventional (CA) and Barren Land (BL). The details of the soil samples, the region’s soil type and the history of the land have been provided in Table 1 . Here we have analyzed the fungal profiles in these plots. The soil chemical profiling for each of the samples was carried out as discussed in Singh et. al., 2023. The soil chemical profiles of the respective soil samples are provided in Table 2 . View this table: View inline View popup Table 1. Soil sampling, classification, agricultural practices and history. View this table: View inline View popup Download powerpoint Table 2: Chemical Parameters of the Soil Samples DNA isolation, Quality check and Sequencing Soil DNA isolation was performed using the DNeasy Power soil kit, following the manufacturer’s protocol. Quality check for the isolated DNA was done using Nanodrop, by estimating A 260 /A 280 ratio. These steps are discussed in detail in Singh et. al., 2023. This was followed by internal transcriber spacing (ITS) based sequencing, for fungal metagenomic analysis. Sequencing was done using Illumina MiSeq platform (Eurofins Genomics India Pvt. Ltd., Bangalore, India). ITS rRNA gene amplification was done using the following primers: ITS 1F (5’-GCATCGATGAAGAACGCAGC-3’) and ITS 2R (5’-TCCTCCGCTTATTGATATGC-3’). Metagenomic Analysis and Statistics The obtained sequences were subjected to metagenomics analysis using QIIME 2 pipeline ( Bolyen et al., 2019 ). This pipeline uses the DADA2 algorithm for the crucial de-noising step, which aids in filtering and trimming reads that do not meet the quality requirements. This is followed by identification of amplicon sequence variants (ASVs). These ASVs are then annotated using a trained classifier model to assign taxonomy. Following this, a thorough literature review was conducted to analyze and understand the results obtained and identify relevant taxa. Statistical analysis was performed on these using unpaired t-tests and one-way ANOVA using Python Programming Language. Functional analysis: FUNGuild FUNGuild v1.1 was used to investigate the function of fungal communities in soil samples ( Nguyen et. al., 2016 ). Twelve functional guilds, namely, ectomycorrhizal fungi, fungal endophytes, wood saprotrophs, plant saprotrophs, dung saprotrophs, undefined saprotrophs, animal pathogens, plant pathogens, algal parasites, animal parasites, fungal parasites and lichen parasites were identified. These were classified according to three trophic modes (pathotrophs, saprotrophs and symbiotrophs). Three confidence ranks, “possible”, “probable”, and “highly probable”, were assigned by the tool. One-way ANOVA was performed using python programming to assess the effect of regenerative agriculture on the relative abundance of OTUs inferred using FUNGuild. Results Metagenomics ITS metagenomics studies were carried out for all 14 soil samples given in Table 1 . The sequence information for the samples can be found in NCBI with the project ID PRJNA912401 (accession numbers SRR28254635 to SRR28254648). A total of 4,656,196 raw sequence reads were obtained from the metagenomic library generated using Illumina. This ranged from 257,444 to 438,222 reads per sample. These were processed to remove primers and other noise and chimeras, to obtain 3,382,950 high quality reads. Fungal taxonomy up to the genus level have been considered for this study. Taxonomic analysis revealed 3907 OTUs in the samples. Abundance of Fungal Populations In vegetable RA plots we found a relatively higher abundance of fungal OTUs (Reg-8VP: 11097, Reg-3VP: 9569, Reg-1VA: 8901, Reg-10VP: 9569 and Reg-12VA: 13707) as compared to that in CA (Con-VP1: 5597 and Con-VP2: 9235) and BL (BL-Euc: 8447 and BL: 9630) ( Figure 1A ). Whereas the fungal OTUs in RA finger millet plots were found to be lesser or equivalent to the CA plots ( Figure 1B ). Download figure Open in new tab Figure 1: Total fungal abundance in A) Vegetable (RA, CA) and BL plots; and in B) Finger millet (RA, CA) and BL plots Abundance of Beneficial Fungi The finger millet and vegetable RA plots were compared with their respective CA controls and BL negative controls for the abundance of beneficial fungi known in literature. Here we have considered the well characterized Plant Growth Promoting Fungi (PGPFs), endophytic fungi and ectomycorrhizal fungi which are well known beneficial genera. Based on the identified taxa in our samples the PGPFs considered in this study include - Cladosporium , Aspergillus , Talaromyces , Inocybe , Psathyrella , Rhizophlyctis , Conidiobolus , Mortierella , Phoma and Pseudorobillarda ( Hossain & Sultana, 2020 ; Adedayo & Babalola, 2023 ; Li et al., 2024 ; Halifu et al., 2021 ; El-Katatny, 2010 ; Gałązka et al., 2020 ). Endophytes identified were Acrocalymma , Agrocybe , Clitopilus , Cunninghamella , Torula and Subulicystidium ( Fite et al., 2023 ; Khasim et al., 2020 ; Atiphasaworn et al., 2017 ; Syamsia et al., 2021 ; Ma et al., 2023 ), while the ectomycorrhizal fungi were Pisolithus and Scleroderma ( Singh et al., 2019 ) ( Figure 2 ). Download figure Open in new tab Figure 2: Abundance (Number of Reads) of beneficial fungi across RA, CA and BL plots with A) Comparing Finger millet plots and B) Comparing Vegetable plots Each of the three RA finger millet plots were found to contain specific beneficial fungal genera - Rhizophlyctis (0.01%) in Reg-8RA, Acrocalymma ( 0.02% ) in Reg-7RA and Agrocybe (0.18%) in Reg-1RA respectively, which were absent in the finger millet CA and BL plots ( Figure 2A ). In addition, Reg-8RA also contained Cladosporium (0.05%) and Mortierella (0.02%), while Reg-7RA also showed presence of Cladosporium (0.02%) and Psathyrella ( 0.02% ). Interestingly, the BL-Euc (Eucalyptus) soil showed highest levels of beneficial fungal genera - Scleroderma ( 0.03% ) , Clitopilus ( 0.14% ) and Inocybe (0.18%). Among the vegetable RA plots, the one carrying out regenerative agriculture for 12 years, Reg-12VA showed enhanced representation of beneficial fungal genera - Mortierella (0.02%), Phoma (0.06%), Torula (0.01%) and Cladosporium (0.05%) ( Figure 2B ). Abundance of Pathogenic Fungi Simultaneously the occurrence and abundance of plant pathogenic fungal genera, chosen based on their occurrence across the samples - Pyrenochaetopsis ( Kwaśna et al., 2021 ), Deniquelata ( Ariyawansa et al., 2013 , Devadatha et al., 2018 ), Periconia ( Gunasekaran et al., 2021 ), Alternaria ( Tozlu et al., 2018 , Pinto & Patriarca, 2016), Bipolaris ( Al-Sadi, 2021 , Aslam et al., 2021 , Naik et al., 2016 ), Microdochium ( Gagkaeva et al., 2020 ), Waitea ( Vojvodić et al., 2022 , Vojvodić et al., 2021 ), Curvularia ( Aguas et al., 2017 , Wei et al., 2022 ), Fusarium ( Rampersad, 2020 , Blanco & Aveling, 2018), Ceratobasidium ( Li et al., 2024 , De Melo et al., 2018 ) and Rigidoporus ( Chaiharn et al., 2019 , Mahendran et al., 2021 ) was explored and compared in RA, CA and BL plots ( Figure 3 ). Download figure Open in new tab Figure 3: Abundance (Number of Reads) of plant pathogenic fungi across RA and CA vegetable plots and BL plots in A) Finger millet and B) Vegetable plots The RA finger millet plots showed an absence of several of the plant pathogenic fungal genera. Reg-8RA was found to contain Bipolaris (0.03%) and Microdochium (0.02%), while Reg-7RA had only Microdochium (0.01%) and Reg-1RA contained none of the plant pathogenic genera. Clearly, the finger millet RA (0.05%, 0.01% and 0%) plots were found to contain fewer of the plant pathogenic fungal genera compared to finger millet CA (0.06% and 0.09%) and BL (0.19% and 0.11%) plots. The well-known plant pathogen genus Bipolaris in finger millet CA plots was found to be (0.05% and 0.06%) and in the BL plots (0.04% and 0.06%) ( Figure 3A ). Statistical analysis (one-way ANOVA) was done which revealed that there is significant decrease in the total abundance of pathogenic fungi in case of finger millet (p-value = 0.0397) In the case of vegetables, the total pathogenic fungal genera in each RA plot (0.02%, 0.01%, 0.22%, 0% and 0.03%) were found to be mostly lesser than that in the BL plots (0.19% and 0.11%). However, the vegetable CA plots did not contain any of the pathogenic fungal genera that we analyzed for this study. Interestingly, none of the vegetable RA or CA plots had Bipolaris or Pyrenochaetopsis while the BL plots contained (0.04% and 0.06%) Bipolaris and (0.04% and 0.03%) Pyrenochaetopsis ( Figure 3B ). Statistical analysis revealed that abundance of the pathogenic fungal genera Bipolaris and Pyrenochaetopsis , have decreased significantly in RA plots both in case of finger millets (p = 0.0355 and p = 0.00077) and vegetables (p= 0.00195 and 0.000017) for Bipolaris and Pyrenochaetopsis respectively. Functional Analysis The identified fungal taxa were classified into ecological-guilds (trophic modes) using the FUNGuild tool to understand their relative distribution patterns in the various agricultural systems versus barren plots. The taxa could be classified into five trophic modes - pathotroph-saprotroph-symbiotrophs, pathotroph-saprotrophs, saprotrophs, saprotroph-symbiotrophs and symbiotrophs. Finger millet RA samples were found to be composed of 71 % saprotrophs, 19% pathotroph-saprotrophs and 10% saprotroph-symbiotrophs, whereas the CA comprised 70% pathotroph-saprotrophs, 14% saprotrophs, 12% saprotroph-symbiotrophs and 4% symbiotrophs and BL had 58% pathotroph-saprotrophs, 38% symbiotrophs, and 4% pathotroph-saprotroph-symbiotrophs ( Figure 4A ). The RA plots show a higher abundance of saprotrophs and lesser levels of pathotroph-saprotrophs while both CA and BL plots are observed to have greater relative abundance of the latter. Download figure Open in new tab Figure 4: Relative abundance of trophic modes in Regenerative agriculture (RA), Conventional agriculture (CA) and Barren land (BL) plots in A) Finger millet and B) Vegetable In vegetables, the RA samples had 37% pathotroph-saprotrophs, 31% pathotroph-saprotroph-symbiotrophs, 16% saprotrophs and 16% saprotroph-symbiotrophs, while the CA was found to comprise of 100% saprotrophs and BL contained 58% pathotroph-saprotrophs, 38% symbiotrophs, and 4% pathotroph-saprotroph-symbiotrophs ( Figure 4B ). In vegetable RA plots although all five trophic modes were present the significant change observed was the additional assemblage of saprotroph-symbiotrophs which is absent in both vegetable CA and BL plots. Statistical analysis of relative abundance of each trophic mode between the samples in A and B ( Figure 4 ) at 95% level of significance revealed the following. In the case of finger millet plots, p-values of 0.340, 0.055, 0.477, 0.487 and 0.128 were obtained for the trophic modes Pathotroph-Saprotroph-Symbiotroph, Pathotroph-Saprotroph, Saprotroph, Saprotroph-Symbiotroph and Symbiotroph respectively. In the case of vegetable plots, p-values were 0.402, 0.002, 0.577, 0.314 and 0.034 for the trophic modes Pathotroph-Saprotroph-Symbiotroph, Pathotroph-Saprotroph, Saprotroph, Saprotroph-Symbiotroph and Symbiotroph respectively. Thus, in vegetable plots (RA and CA) versus BL plots significant differences were observed in case of pathotroph-saprotrophs and symbiotrophs. Although these results largely indicate that there is not much significant difference in the trophic modes assembly among RA, CA and BL, this could be attributed to the smaller sample size used in this study. Aside from the trophic modes, FUNGuild was also used to assign functional groups or guilds to the OTUs. This revealed that among finger millet RA samples, most fungi belonged to saprotrophic guilds (plant saprotrophs, wood saprotrophs, undefined saprotrophs and dung saprotrophs) along with others like endophytes, algal parasites and plant pathogens. Plant pathogens were absent in Reg-7RA and Reg-1RA and comprised a lesser fraction in Reg-8RA (17.39%). Contrary to this the CA plots apart from hosting saprotrophs (undefined saprotrophs and plant saprotrophs), plant pathogens and endophytes, also contained animal parasites and animal pathogens ( Figure 5A ). More importantly, Reg-7RA showed higher abundance (50%) of wood saprotrophs. Download figure Open in new tab Figure 5: Relative abundance of functional guilds in respective RA, CA and BL plots A) Finger millets and B) Vegetable plots Among vegetables, a large share of the RA samples consisted of saprotrophic fungi (wood, undefined and plant saprotrophs). We also observed an enhanced representation of endophytes in RA vegetable plots. In addition, RA vegetable plots also contain plant pathogens and lichen parasites. In contrast, CA (vegetable) and BL had saprotrophs, plant pathogens, fungal parasites, animal parasites, animal pathogens and ectomycorrhizal fungi ( Figure 5B ). Regenerative agriculture samples in both cases have a significant percentage of saprotrophic guilds, including plant saprotrophs. Discussion This is the first ever report from India that has attempted to study the soil fungal populations in regenerative agriculture versus conventional agriculture, and barren and eucalyptus-containing land. Like several other studies on fungal community profiling, we also did not find a consistent pattern of fungal assemblages with agricultural practices ( Hannula, et.al., 2021 ; Sommermann, et.al., 2018 ; Schneider, et.al., 2010 ). Also, with many fungal species still not identified, the characterization of fungal community structures is extremely difficult. Moreover, with almost negligible information available on fungi in Indian soils, it is essential to analyze and qualify soil health and ecosystems based on fungal populations in India. The vegetable RA plots interestingly showed higher fungal abundance compared to the vegetable CA plots. This may be due to the healthier soil management practices and organic supplementations used in RA contributing to a thriving fungal flora. In addition, the use of pesticides and herbicides in conventional agriculture has been suggested to cause unintended harmful effects on soil microorganisms (Steiner, et.al., 2024). Furthermore, the vegetable RA plot Reg-12VA, ( Table 1 ) observed the highest fungal population. Reg-12VA also contained the highest levels of beneficial fungi among all the RA plots. These could be attributed to 12 years of natural farming practiced here with heavy input of green farm mulch which retains moisture and forms a rich carbon resource supporting the growth of decomposers especially fungi ( Iqbal, et. al., 2020 ). Studies also show that mulching and compost amendments reduce proliferation of pathogenic fungal strains ( Entry, et. al., 2005 ; Neher, et. al., 2022; Vida, et. al., 2020 ; Richardville, et. al., 2022 ; Blaya, et. al., 2016 ). Mulch accumulates microbes with cellulose degrading properties which also act on cell walls of fungal pathogens. Conforming with these findings we also report that both vegetable and finger millet RA plots practicing for at least 3 years and more had reduced numbers of pathogenic fungal genera. Both the finger millet and vegetable RA plots showed significantly lower levels of well-known plant pathogens Bipolaris and Pyrenochaetopsis . In vegetable CA plots a complete absence of all the assessed pathogenic genera was observed, suggesting either that the usage of farmyard manure promotes the growth of pathogen suppressing strains or the use of pesticides and herbicides by these farms has contributed to their elimination. Functional analysis of the fungal communities revealed an interesting, enhanced occurrence of saprotrophs in RA finger millet plots while CA finger millet and BL plots showed highest representation of pathotroph-saprotrophs. Literature shows that soil amendments with rich sources of organic matter stimulate proliferation of saprotrophic fungi ( Clocchiatti, et. al., 2021 ). Regenerative agriculture practices using farmyard manure, vermicompost, cow dung, leaves and green mulch as used by the plots Reg-1RA, Reg-7RA and Reg-8RA provide the necessary organic matter that enhances saprotrophic fungal growth. Our findings suggest that the proliferating saprotrophs enrich the soil for nutrients in plant assimilable forms by decomposing organic matter. Additionally, their overactive cellulase enzymes non-specifically also digest the pathotroph-saprotrophs leading to their simultaneous decline ( Nam, et. al., 2023 ). Thus, organic input agriculture provides us with the desired dual advantage of enhancing soil nutrients while concomitantly reducing pathogenic strains. In contrast, the vegetable RA plots showed higher fractions of pathotroph-saprotrophs and pathotroph-saprotroph-symbiotrophs while the CA plot had the desired 100% saprotroph composition. Here we construe that since the vegetable CA plots are using organic supplementation alongside the pesticide and herbicides, the latter are damaging the fungal populations, but the organic supplements are allowing for the revival of the saprotrophs. On the contrary, in the vegetable RA plots the fungal populations are in a dynamically evolving phase which keeps changing with crops and organic supplementations and may take several years to reach a mature and more organized structure. To understand this, it is necessary to study well evolved farms that have been carrying out regenerative practice for at least 20 - 25 years. Somehow, none of the assayed farms showed the presence of Arbuscular Mycorrhizal Fungi (AMFs) which are known to be significant supporters of plant growth and productivity. AMFs also form an important subset of the saprotrophic fungal guild. Studies suggest that among several factors that dictate enhancement of the plant’s synergistic association with AMFs are - (i) limiting nitrogen and phosphorus conditions; (ii) increased root secretion of strigolactones and flavonoids and (iii) vicinity to tree populations ( Shantz, et. al., 2016 ; Della Mónica, et. al., 2023 ; Dierks, et. al., 2021 ). The relatively scant population of trees around most of the studied agricultural plots might have contributed to the noted absence of AMFs in our study. This stirs up the consequential debate - whether agroforestry should be the recommended practice of choice for regenerative agriculturists. Another noteworthy point was that the BL-Euc plot contained the highest levels of beneficial as well as pathogenic fungal genera. Greater association of fungi around trees is a common phenomenon, as trees provide an abundant carbon resource in the form of dead wood, dried leaves etc ( Manici, et. al., 2023 ; Dierks, et. al., 2021 ). In our previous report on bacteria in these same plots ( Singh, et. al., 2023 ), we found that bacterial diversity had pronouncedly increased in natural agriculture farms and was directly proportional to the number of years of RA implementation. Although fungal abundance was found to increase in vegetable RA plots since we could not see a similar trend in finger millet plots, we are unable to confidently conclude this finding. This points to the need for many more experiments with larger sample sizes to verify this result. In contrast to the bacterial study where we could see a remarkable accumulation of PGPRs in RA plots, fungal study showed a decline in pathogenic fungal strains in RA plots. It is evident that fungi and bacterial populations respond differently to agricultural practices, however we can conclude that RA brings along a healthy improvement in their compositions thus enhancing the vitality of crops and their productivity. Author contributions IS was involved in planning the experiment, collecting soil samples, DNA isolation, data analysis as well as writing the manuscript. PN has done the complete ITS metagenomic data analysis for the fungal community structure in plots and wrote the Materials and Methods and Results Section with the Figures in this manuscript. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgements This research is part of the SHEFS - an interdisciplinary research partnership, forming part of the Wellcome Trust’s funded Our Planet, Our Health programme, with the overall objective to provide novel evidence to define future food systems policies to deliver nutritious and healthy foods in an environmentally sustainable and socially equitable manner. This research was funded by the Wellcome Trust through the Sustainable and Healthy Food Systems (SHEFS) Project (Grant number-205200/Z/16/Z). The authors would like to thank ATREE for facilitating the initial research. References 1. ↵ Adedayo , A.A. and Babalola , O.O ., 2023 . Fungi that promote plant growth in the rhizosphere boost crop growth . Journal of Fungi , 9 ( 2 ), p. 239 . OpenUrl 2. ↵ Aguas , Y. , Hincapie , M. , Fernández-Ibáñez , P. and Polo-López , M.I ., 2017 . Solar photocatalytic disinfection of agricultural pathogenic fungi (Curvularia sp.) in real urban wastewater . Science of the total environment , 607 , pp. 1213 – 1224 . OpenUrl 3. ↵ Al-Sadi , A.M ., 2021 . Bipolaris sorokiniana-induced black point, common root rot, and spot blotch diseases of wheat: A review . Frontiers in cellular and infection microbiology , 11 , p. 584899 . OpenUrl 4. ↵ Ariyawansa , H.A. , Maharachchikumbura , S.S. , Karunarathne , S.C. , Chukeatirote , E. , Bahkali , A.H. , Kang , J.K. , Bhat , D.J. and Hyde , K.D ., 2013 . Deniquelata barringtoniae gen. et sp. nov., associated with leaf spots of Barringtonia asiatica . Phytotaxa , 105 ( 1 ), pp. 11 – 20 . OpenUrl 5. ↵ Aslam , H.M.U. , Naveed , K. , Hussain , S.I. , Shakeel , Q. , Ashraf , W. , Anwaar , H.A. , Raza , M.M. , Sarfraz , S. and Tariq , I ., 2021 . First report of brown leaf spot of rice caused by Bipolaris zeicola in Pakistan . Plant Disease , 105 ( 1 ), p. 212 . OpenUrl 6. ↵ Atiphasaworn , P. , Monggoot , S. , Gentekaki , E. , Brooks , S. and Pripdeevech , P ., 2017 . Antibacterial and antioxidant constituents of extracts of endophytic fungi isolated from Ocimum basilicum var. thyrsiflora leaves . Current microbiology , 74 , pp. 1185 – 1193 . OpenUrl 7. ↵ Bach , E.M. , Ramirez , K.S. , Fraser , T.D. and Wall , D.H ., 2020 . Soil biodiversity integrates solutions for a sustainable future . Sustainability , 12 ( 7 ), p. 2662 . OpenUrl 8. Blanco , R. and Aveling , T.A.S. , 2016 , September. Seed-borne Fusarium pathogens in agricultural crops . In VII International Symposium on Seed, Transplant and Stand Establishment of Horticultural Crops-SEST2016 1204 (pp. 161 – 170 ). 9. ↵ Blaya , J. , Marhuenda , F.C. , Pascual , J.A. and Ros , M ., 2016 . Microbiota characterization of compost using omics approaches opens new perspectives for Phytophthora root rot control . PloS one , 11 ( 8 ), p. e0158048 . OpenUrl CrossRef 10. ↵ Bolyen , E. , Rideout , J.R. , Dillon , M.R. , Bokulich , N.A. , Abnet , C.C. , Al-Ghalith , G.A. , Alexander , H. , Alm , E.J. , Arumugam , M. , Asnicar , F. and Bai , Y ., 2019 . Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 . Nature biotechnology , 37 ( 8 ), pp. 852 – 857 OpenUrl CrossRef PubMed 11. ↵ Chaiharn , M. , Sujada , N. , Pathom-Aree , W. and Lumyong , S ., 2019 . Biological control of Rigidoporus microporus, the cause of white root disease in rubber using PGPRs in vivo . Chiang Mai J. Sci , 46 ( 5 ), pp. 850 – 866 . OpenUrl 12. ↵ Clocchiatti , A. , Hannula , S.E. , Hundscheid , M.P. , klein Gunnewiek , P.J. and de Boer , W. , 2021 . Stimulated saprotrophic fungi in arable soil extend their activity to the rhizosphere and root microbiomes of crop seedlings . Environmental Microbiology , 23 ( 10 ), pp. 6056 – 6073 . OpenUrl 13. ↵ Costantini , E.A. and Mocali , S ., 2022 . Soil health, soil genetic horizons and biodiversity# . Journal of Plant Nutrition and Soil Science , 185 ( 1 ), pp. 24 – 34 . OpenUrl 14. ↵ De Gannes , V. , Bekele , I. , Dipchansingh , D. , Wuddivira , M.N. , De Cairies , S. , Boman , M. and Hickey , W.J. , 2016 . Microbial community structure and function of soil following ecosystem conversion from native forests to teak plantation forests . Frontiers in microbiology , 7 , p. 1976 . OpenUrl 15. ↵ De Melo , M.P. , Matos , K.S. , Moreira , S.I. , Silva , F.F. , Conceição , G.H. , Nechet , K.L. , Halfeld-Vieira , B.A. , Beserra Junior , J.E. , Ventura , J.A. , Costa , H. and Furtado , E.L. , 2018 . Two new Ceratobasidium species causing white thread blight on tropical plants in Brazil . Tropical plant pathology , 43 , pp. 559 – 571 . OpenUrl 16. ↵ Della Mónica , I.F. , Vignale , M.V. , Scervino , J.M. , Iannone , L.J. and Novas , M.V ., 2023 . Role of fungal endophytes on mycorrhizal-plant association and its impact on plant fitness . In Microbial Endophytes and Plant Growth (pp. 117 – 136 ). Academic Press . 17. ↵ Devadatha , B. , Sarma , V.V. , Ariyawansa , H.A. and Jones , E.G. , 2018 . Deniquelata vittalii sp. nov., a novel Indian saprobic marine fungus on Suaeda monoica and two new records of marine fungi from Muthupet mangroves, East coast of India . 18. ↵ Dierks , J. , Blaser-Hart , W.J. , Gamper , H.A. , Nyoka , I.B. , Barrios , E. and Six , J ., 2021 . Trees enhance abundance of arbuscular mycorrhizal fungi, soil structure, and nutrient retention in low-input maize cropping systems. Agriculture , Ecosystems & Environment , 318 , p. 107487 . OpenUrl 19. ↵ Doehlemann , G. , Ökmen , B. , Zhu , W. and Sharon , A ., 2017 . Plant pathogenic fungi . The fungal kingdom , pp. 701 – 726 . 20. ↵ Du , Z.Y. , Zienkiewicz , K. , Vande Pol , N. , Ostrom , N.E. , Benning , C. and Bonito , G.M ., 2019 . Algal-fungal symbiosis leads to photosynthetic mycelium . Elife , 8 , p. e47815 . OpenUrl CrossRef 21. ↵ El-Katatny , M.H ., 2010 . Enzyme production and nitrogen fixation by free, immobilized and co-immobillzed inoculants of Trichoderma harzianum and Azospirillum brasilense and their possible role in growth promotion of tomato . 22. ↵ Entry , J.A. , Strausbaugh , C.A. and Sojka , R.E ., 2005 . Compost amendments decrease Verticillium dahliae infection on potato . Compost science & utilization , 13 ( 1 ), pp. 43 – 49 . OpenUrl 23. ↵ Fite , T. , Kebede , E. , Tefera , T. and Bekeko , Z ., 2023 . Endophytic fungi: versatile partners for pest biocontrol, growth promotion, and climate change resilience in plants . Frontiers in Sustainable Food Systems , 7 , p. 1322861 . OpenUrl 24. ↵ Frąc , M. , Sas-Paszt , L. and Sitarek , M ., 2023 . Changes in the Mineral Content of Soil following the Application of Different Organic Matter Sources . Agriculture , 13 ( 6 ), p. 1120 . OpenUrl 25. ↵ Gagkaeva , T.Y. , Orina , A.S. , Gavrilova , O.P. and Gogina , N.N ., 2020 . Evidence of Microdochium fungi associated with cereal grains in Russia . Microorganisms , 8 ( 3 ), p. 340 . OpenUrl 26. ↵ Gałązka , A. , Grządziel , J. , Gałązka , R. , Gawryjołek , K. , Ukalska-Jaruga , A. and Smreczak , B ., 2020 . Fungal community, metabolic diversity, and glomalin-related soil proteins (GRSP) content in soil contaminated with crude oil after long-term natural bioremediation . Frontiers in Microbiology , 11 , p. 572314 . OpenUrl 27. ↵ Gunasekaran , R. , Janakiraman , D. , Rajapandian , S.G.K. , Appavu , S.P. , Venkatesh , P.N. and Prajna , L ., 2021 . Periconia species-An unusual fungal pathogen causing mycotic keratitis . Indian Journal of Medical Microbiology , 39 ( 1 ), pp. 36 – 40 . OpenUrl 28. ↵ Halifu , S. , Deng , X. , Zhang , J. , Xia , J. , Song , X. and Song , R ., 2021 . Promoting the Growth of Pinus sylvestris var. mongolica Seedlings and Improving Rhizosphere Fungal Community Structure through Interaction between Trichoderma and Ectomycorrhizal Fungi . Research in Ecology , 3 ( 2 ), pp. 63 – 79 . OpenUrl 29. ↵ Hannula , S.E. , Di Lonardo , D.P. , Christensen , B.T. , Crotty , F.V. , Elsen , A. , van Erp , P.J. , Hansen , E.M. , Rubæk , G.H. , Tits , M. , Toth , Z. and Termorshuizen , A.J. , 2021 . Inconsistent effects of agricultural practices on soil fungal communities across 12 European long-term experiments . European Journal of Soil Science , 72 ( 4 ), pp. 1902 – 1923 . OpenUrl 30. ↵ Hossain , M.M. and Sultana , F ., 2020 . Application and mechanisms of plant growth promoting fungi (PGPF) for phytostimulation . Organic agriculture , 1 , p. 31 . OpenUrl 31. ↵ Hyde , K.D ., 2022 . The numbers of fungi . Fungal Diversity , 114 ( 1 ), pp. 1 – 1 . OpenUrl 32. ↵ Iqbal , R. , Raza , M.A.S. , Valipour , M. , Saleem , M.F. , Zaheer , M.S. , Ahmad , S. , Toleikiene , M. , Haider , I. , Aslam , M.U. and Nazar , M.A ., 2020 . Potential agricultural and environmental benefits of mulches—a review . Bulletin of the National Research Centre , 44 , pp. 1 – 16 . OpenUrl 33. ↵ Khasim , S.M. , Long , C. , Thammasiri , K. and Lutken , H . eds., 2020 . Medicinal plants: biodiversity, sustainable utilization and conservation . Singapore : Springer . 34. ↵ Kwaśna , H. , Szewczyk , W. , Baranowska , M. , Gallas , E. , Wiśniewska , M. and Behnke-Borowczyk , J ., 2021 . Mycobiota associated with the vascular wilt of poplar . Plants , 10 ( 5 ), p. 892 . OpenUrl 35. ↵ Li , D. , Jin , X.H. , Li , Y. , Wang , Y.C. , He , H.Y. and Zhang , H.B ., 2024 . Fungal communities associated with early immature tubers of wild Gastrodia elata . Ecology and Evolution , 14 ( 2 ), p. e11004 . OpenUrl 36. Li , S.N. , Jiang , S.C. and Zhang , W.M ., 2024 . First Report of Postharvest Fruit Rot of Loquat (Rhaphiolepis loquata) Caused by Ceratobasidium sp. in China . Plant Disease , 108 ( 2 ), p. 533 . OpenUrl 37. ↵ Ma , N. , Yin , D. , Liu , Y. , Gao , Z. , Cao , Y. , Chen , T. , Huang , Z. , Jia , Q. and Wang , D ., 2023 . Succession of endophytic fungi and rhizosphere soil fungi and their correlation with secondary metabolites in Fagopyrum dibotrys . Frontiers in Microbiology , 14 , p. 1220431 . OpenUrl 38. ↵ Mahendran , T.R. , Thottathil , G.P. , Surendran , A. , Nagao , H. and Sudesh , K ., 2021 . Biocontrol potential of Aspergillus terreus, endophytic fungus against Rigidoporus microporus and Corynespora cassiicola, pathogens of rubber tree . Archives of Phytopathology and Plant Protection , 54 ( 15-16 ), pp. 1014 – 1032 . OpenUrl 39. ↵ Manici , L.M. , De Meo , I. , Sacca , M.L. , Ceotto , E. , Caputo , F. and Paletto , A. , 2023 . The relationship between tree species and wood colonising fungi and fungal interactions influences wood degradation . Ecological Indicators , 151 , p. 110312 . OpenUrl 40. ↵ Naik , M.R. , Akila , R. and Thiruvudainambi , S ., 2016 . Management approaches for brown spot of rice caused by Bipolaris oryzae . Journal of Farm Sciences , 29 ( 3 ), pp. 370 – 376 . OpenUrl 41. ↵ Nam , B. , Lee , H.J. and Choi , Y.J ., 2023 . Organic farming allows balanced fungal and oomycetes communities . Microorganisms , 11 ( 5 ), p. 1307 . OpenUrl 42. ↵ Nguyen , N. H. , Song , Z. , Bates , S. T. , Branco , S. , Tedersoo , L. , Menke , J. , … & Kennedy , P. G. ( 2016 ). FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild . Fungal Ecology , 20 , 241 – 248 . OpenUrl 43. ↵ Pandit , M.A. , Kumar , J. , Gulati , S. , Bhandari , N. , Mehta , P. , Katyal , R. , Rawat , C.D. , Mishra , V. and Kaur , J ., 2022 . Major biological control strategies for plant pathogens . Pathogens , 11 ( 2 ), p. 273 . OpenUrl 44. Pinto , V.E.F. and Patriarca , A ., 2017 . Alternaria species and their associated mycotoxins . Mycotoxigenic Fungi: Methods and Protocols , pp. 13 – 32 . 45. ↵ Poveda , Jorge , Sandra Díaz-González , María Díaz-Urbano , Pablo Velasco , and Soledad Sacristán . " Fungal endophytes of Brassicaceae: Molecular interactions and crop benefits ." Frontiers in plant science 13 ( 2022 ): 932288 . OpenUrl 46. ↵ Rampersad , S.N ., 2020 . Pathogenomics and management of Fusarium diseases in plants . Pathogens , 9 ( 5 ), p. 340 . OpenUrl 47. ↵ Richardville , K. , Egel , D. , Flachs , A. , Jaiswal , A. , Perkins , D. , Thompson , A. and Hoagland , L ., 2022 . Leaf mold compost reduces waste, improves soil and microbial properties, and increases tomato productivity . Urban Agriculture & Regional Food Systems , 7 ( 1 ), p. e20022 . OpenUrl 48. ↵ Schneider , S. , Hartmann , M. , Enkerli , J. and Widmer , F ., 2010 . Fungal community structure in soils of conventional and organic farming systems . Fungal Ecology , 3 ( 3 ), pp. 215 – 224 . OpenUrl 49. ↵ Schröder , J.J. , Schulte , R.P.O. , Creamer , R.E. , Delgado , A. , Van Leeuwen , J. , Lehtinen , T. , Rutgers , M. , Spiegel , H. , Staes , J. , Tóth , G. and Wall , D.P. , 2016 . The elusive role of soil quality in nutrient cycling: a review . Soil Use and Management , 32 ( 4 ), pp. 476 – 486 . OpenUrl 50. ↵ Selvasekaran , P. and Chidambaram , R ., 2020 . Agriculturally important fungi for crop protection . Agriculturally Important Fungi for Sustainable Agriculture: Volume 2: Functional Annotation for Crop Protection , pp. 1 – 53 . 51. ↵ Shantz , A.A. , Lemoine , N.P. and Burkepile , D.E ., 2016 . Nutrient loading alters the performance of key nutrient exchange mutualisms . Ecology letters , 19 ( 1 ), pp. 20 – 28 . OpenUrl 52. ↵ Singh , D.P. , Gupta , V.K. and Prabha , R. eds., 2019 . Microbial interventions in agriculture and environment: Volume 2: Rhizosphere, microbiome and agro-ecology . Springer Nature . 53. ↵ Singh , I. , Hussain , M. , Manjunath , G. , Chandra , N. and Ravikanth , G ., 2023 . Regenerative agriculture augments bacterial community structure for a healthier soil and agriculture . Frontiers in Agronomy , 5 , p. 1134514 . OpenUrl 54. ↵ Sommermann , L. , Geistlinger , J. , Wibberg , D. , Deubel , A. , Zwanzig , J. , Babin , D. , Schlüter , A. and Schellenberg , I ., 2018 . Fungal community profiles in agricultural soils of a long-term field trial under different tillage, fertilization and crop rotation conditions analyzed by high-throughput ITS-amplicon sequencing . PloS one , 13 ( 4 ), p. e0195345 . OpenUrl CrossRef 55. ↵ Syamsia , S.S. , Abubakar , A.I. , Amanda , A.P.F. , Noerfitryani , N.N. , Iradhatullah , I.R. , Henry , H.K. And Rakhmad , R.A. , 2021 . Combination of endophytic fungal as the Plant Growth-Promoting Fungi (PGPF) on cucumber (Cucumis sativus) . Biodiversitas , 22 ( 3 ), pp. 1194 – 1202 . OpenUrl 56. ↵ Tozlu , E. , Tekiner , N.A.S.I.B.E. , Kotan , R. and Örtücü , S.E.R.K.A.N ., 2018 . Investigation on the biological control of Alternaria alternata . Indian Journal of Agricultural Sciences , 88 ( 8 ). OpenUrl 57. ↵ Vida , C. , de Vicente , A. and Cazorla , F.M. , 2020 . The role of organic amendments to soil for crop protection: induction of suppression of soilborne pathogens . Annals of Applied Biology , 176 ( 1 ), pp. 1 – 15 . OpenUrl 58. ↵ Vojvodić , M. , Tanović , B. , Mitrović , P. , Vico , I. and Bulajić , A ., 2021 . Waitea circinata var. zeae causing root rot of cabbage and oilseed rape . Plant Disease , 105 ( 4 ), pp. 787 – 796 . OpenUrl 59. ↵ Vojvodić , M. , Tanović , B. , Mitrović , P. , Vico , I. and Bulajić , A. , 2022 . Pathogenic potential of Waitea circinata var. zeae . In Book of Abstracts, 11th International Symposium of Agricultural Sciences “AgroReS 2022", 26-28 May 2022, Trebinje, Bosnia and Herzegovina (pp. 83 – 84 ). Banja Luka: University of Banja Luka, Faculty of Agriculture . 60. ↵ Wei , T. , Luo , M. , Zhang , H. , Jia , W. , Zeng , Y. and Jiang , Y ., 2022 . Curvularia verruculosa as new causal pathogen of common bean leaf spot disease in China . Crop Protection , 162 , p. 106091 . OpenUrl View the discussion thread. Back to top Previous Next Posted August 15, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. 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