Wheat Endophytes And Their Beneficial Functions

Endophytesplay a vital role in growing healthy crops.In this study the diversity of endophytic nitrogen-fixing bacteria in wheat was studied;a large number of culturable wheat endophytes?618?were isolated and identified,and their potential growth-promoting,antimicrobial and antidiabetic properties were studied.1.Fifteen wheat plant samples of different growth stages were collected from Shaanxi,Shanxi,Hebei and Beijing.The main purpose of the research was to study endophytes diversity.600 strains of bacteria,2 strains of actinomycetes and 16 strains of fungi were isolated from roots,stems and leaves of wheat plants.After their molecular identification,the isolated bacteria were found to belong 23 genera?Achromobacter,Bacillus,Citrobacter,Enterobacter,Flavobacterium,Flexibacter,Klebsiella,Paenibacillus,Kosakonia,Microbacterium,Ochrobactrum,Providencia,Pseudomonas,Rahnella,Rheinheimera,Rhizobium,Serratia,Sphingobacterium,Stenotrophomonas,Cedaceae,Sphingobium,Xenophilus and Variovorax?and 58 species.Among them,12 genera showed the presence of nif H gene?22 species?.Two strains of actinomycetes were identified as Streptomyces;the isolated fungi identified as 15 differentspecies.2.The diversity of wheat endophytic nitrogen-fixing bacteria exhibited variation with the growing areas.From Shaanxi wheat samples?581C,581T,584C and 584T?total 17 genera,33 species were isolated,among them 8 genera exhibited the presence of nif H gene?14 species?.Total 11 genera and 19species of bacteria were isolated from Shanxi samples,among them7 genera and 13 species confirmed nif H gene.From Hebei samples 5 generawere isolated?5 species?,all of themcontained nif H gene.Total 6 genera were collected from Beijing samples,all of them showed the presence of nif H gene?6species?.Variovorax,Streptomyces,Rhizobium,Sphingobium,Kosakonia,Rahnellaand Providenciawere only found in Shaanxi samples.On the contrary,Xenophilus,Serratia,Citrobacterand Bacillus were only isolated from Shanxi samples while Cedaceae was only foundin Beijing samples.3.This study revealed that the fertilizer typealso affected the diversity of nitrogen fixing bacteria.From the biofertilizer treated plants 19 bacteria genera and35 species were isolated,among them 12genera and 22 speciescontained nif H gene.On the contrary 16 genera and 30 species were isolated from the chemical fertilizer treated samples,among them only 7genera showed the existence of nif H gene.It should be noted here that chemical fertilizer treated samples showed more unique genera?Rhizobium,Serratia,Streptomyces,Sphingobium,Xenophilus and Variovorax?than the biofertilizers treated plant samples?Citrobacterand Cedaceae?.4.The diversity of endophytic nitrogen-fixing bacteriawas also significantly observed in different parts of plant samples.Total 20 bacterial genera and 38 specieswere isolated from root,among them 12genera and 22 speciesexhibitednif H gene existance.Whereas 10 genera and 25 species were isolated from stem,among them 7 genera and 16 speciesshowed the presence of nif H gene.On the other hand,9genera and 19 speciesof bacteria were isolatedfrom leaf,where 5 genera and 8 species confirmed the existence of nif Hgene.The highest diversity of endophytic nitrogen fixing bacteria was observed in root while lowest in leaf.The most unique genera?8?were found in root?Cedaceae,Bacillus,Citrobacter,Kosakonia,Providencia,Rhizobium,SphingobiumandSerratia?,moderate in leaf?Variovorax and Streptomyces?and lowest in stem?Flexibacter?.5.The diversity of nitrogen fixing bacteria was also studied in different growth stages of wheat plants.At booting stages samples were collected from Shaanxi and Shanxi provincesand 20 generaand36 species were isolated,among them 9 genera and 17 species exhibited the presence of nif H gene.On the contrary wheat seedlingswere collected from Beijing and Hebei provinces from which only 6 genera and 6 species were isolated and all of them exhibited the presence of nif H gene.It was also observed that diversity was higher in booting stages samples than the seedlings whereas Citrobacterwas found inbooting stages samples and Cedaceae was found in seedling stage as a unique genus.6.As culture dependent method was not enough to find out the actual population of endophytic nitrogen fixing bacteria in wheat plant samples so 16S rRNAand nif H high-throughput sequencing techniques were carried out.The former technique showed the presence of near about 500 genera of bacteria in the studied samples,among them the top generawere Sinorhizobium,Promicromonospora,Pantoea,Massiliaand Klebsiella;nif H high-throughput sequencing result showed the presence of around 100 genera in wheat samples,among them the major were Pseudacidovorax,Sinorhizobium,Variovorax,Leptothrix,Rubrivivax,Skermanella,Pelomonas,Burkholderia,Kluyvera,Kosakonia,Bradyrhizobium and Lentzea.In comparison to culture dependent method,culture independent method showed higher diversity of endophytic nitrogen fixing and nif H containing bacteria.Rahnella,Xenophilus,Serratia,Citrobacter,Flexibacterand Cedaceae were only isolated by traditional methodand were not identified by HTS;whereas most bacteria which were detected by HTS were not possible to isolate by culture dependent method.7.For growth promoting and abiotic stress tests 72 bacteria,2 actinomycetes and 15 fungi were selected from the isolated endophytes.In growth promoting tests,a number of endophytes exhibited quite good nitrogenase(18.16±0.82⁸78.77±0.89nano mol ethylene mg^-1protein^-1h),ACC deaminase(3.97±0.02⁰.03±0.011?mol?-KB mg^-1 protein hr^-1),IAA(1.00±0.1⁴9.37±0.005?gml^-1)and PSI?2.17±0.02⁵.16±0.36?activities.More than 80%isolates gave positive result in siderophore,NH₃ and urease tests;but only few of them?13?produced HCN.On the contrary,in abiotic stress tests,Trichoderma strains showed tolerance at high levels of salt?10%?,heavy metals(300?gml^-1)and drought?35%PEG?and exhibited resistanceagainst all the tested antibiotics;Talaromyces funiculosus strain was found to be the most temperature?55⁰C?tolerant isolate.In pot study,among 20 selected endophytes,7 showed promising growth promoting effects?plant height,dry biomass and SPAD value?on both wheat and maize plants compared to the control.8.All the microbial extracts revealed the presence of a number of valuable bioactive components?alkaloids,flavonoids,tannin etc.?and demonstrated antidiabetic response in dose depending manner where fungal extracts were more potent than bacterial one.Aspergillus niger,Penicillium aurantiogriseum,Trichoderma aureovirideshowed antidiabetic potency near to standard acarbose and among them Penicilliumaurantiogriseumexhibited highest percent of inhibition?94.56±0.11?at400?gml^-1 dose.In antimicrobial test,among all extracts Trichoderma inhibitedall the tested pathogenic bacteria and fungi.9.A novel gram variable bacteria,Paenibacillusrhizophilus 7197^Twas isolated from rhizospheric root of wheat;it was found to contain different types of fatty acids(anteiso-C_15:0,C₁₄:_0,C₁₆:₀etc.),polar lipids?DPG,PG,PE etc.?,respiratory quinine MK-7,Meso-DAP acid,4nif H genes and showed high nitrogenase activity(2526.41±34.9 nano mol ethylene mg^-1 protein^-1h).It also showed ACC deaminase(1.53±0.01?mol?-KB mg^-1 protein hr^-1),IAA(26.12±0.004?gml^-1),PSI?2.08±0.36?,NH₃,siderophore activities and exhibited resistanceagainstvarious abiotic stresses[temperature?40⁰C?,salinity?2.5%NaCl?,drought?20%PEG?,heavy metals and antibiotic sensitivity].It also exhibited promising growth promoting effects?plant height,dry biomassand SPAD value?on wheat and maize plants in comparison to control in pot test.It is concluded that the highest diversity of wheat endophytic nitrogen fixing and nif H containing bacteria was found in the samples collected from Shaanxi.The maximum diversity was observed in root part of plants,biofertilizertreated plants and at booting stage.Compared with culture dependent method,culture independent method showed greater endophytic nitrogen fixing and nif H containing bacteria diversity.Wheat endophytes exhibited potential growth promoting and pharmacological activities?antidiabeticandantimicrobial?.Theisolatednovelbacterium,Paenibacillusrhizophilus7197^Tdemonstrated better growth promoting and abiotic stress tolerance activities.Therefore these research findings revealed that wheat endophytes would be beneficial for the development of organic agriculture and pharmacology in future.