POTENTIAL OF FUNGAL ENDOPHYTES TO ANTAGONISE PUCCINIA STRIIFORMIS CAUSING WHEAT YELLOW RUST

Intense exploitation of pesticides has instigated agricultural and environmental hazards. Microbial inoculation is an alternate to pesticides for antagonising pathogens and is an environmental friendly approach. In this study, we evaluated the potential of fungal endophytes of desert plants, to control the yellow rust of wheat ( Triticum aestivum L.) caused by Puccinia striiformis . Four endophytes i.e. Colletotrichum lindemuthianum, Piriformospora indica , Acremonium lolii and Trichoderma viride were selected in vitro , on the basis of their positive impact on the growth of wheat seedlings. Seeds of two rust susceptible wheat genotypes namely Fareed-06 and Shafaq-06 selected by screening experiment against the disease were treated with spore suspensions of these four endophytes separately using randomized complete block design under factorial arrangement. The data concerning area under disease progress curve (AUDPC), final disease severity (FDS), coefficient of infection (CI), thousand grains weight (TGW) and grain yield (GY) was recorded. Results showed that endophytic inoculated susceptible wheat genotypes exhibited the tolerance against the Puccinia striiformis . The endophyte P. indica significantly decreased the final disease severity and area under disease progress curve , resulting an increase in the grain yield 12.2% in the Fareed-06 and Shafaq-06 followed by the endophytes T. viride, C. lindemuthianum and A. lolii as 10.6%, 06.2% and 04.2% respectively. The present study concluded that fungal endophytes are valuable microbes which can be employed to induce tolerance against P. striiformis and yellow rust for better and sustainable wheat production.


INTRODUCTION
Wheat (Triticum aestivum L.) is widely cultivated cereal crop in the majority of the world regions and is the vital and essential staple food of 36% world's population (2 billion people) [1].The food security is highly focused on obtaining more food to fulfil the needs of burgeoning population which can only be accomplished when the production of wheat as well as other cereal crops is increased globally.The wheat crop is often low as contrast to its actual capacity in terms of production and yield because it hit by many of the abiotic and biotic maladies [2].Among biotic factors, diseases badly affect wheat yield in which rusts have caused huge yield losses and significant damage in recent years.Wheat rusts are prevalent throughout the world and its new races are evolving unremittingly day by day and infecting resistant varieties [3].
Yellow rust of wheat caused by basidiomycete fungus Puccinia striiformis is the most significant wheat disease worldwide favoured cool summer, mild winter, long cool and wet spring.Owing to its economic and social losses, it is presently unsympathetic threat to the global food security [4].Susceptible genotypes show the appearance of yellow streaks on the leaves, followed by elongated, bright yellow, small pustules arranged in obvious rows on leaf sheaths, awns and glumes resultantly spores accumulation within the florets and on developing grain.Seed produced from rust damaged crop express low vigour and poor emergence after germination.Wheat yield losses of 10-70% depend on initial infections, susceptibility of the genotypes, inoculum density and its multiplication rate [5].Early infection causes 100% yield loss on susceptible genotypes whereas disease persists to develop during the whole growing season [6].
To minimize the severe wheat yield losses by different pathogenic diseases and abiotic stresses, appropriate strategies might be adopted.Numerous scientists and researchers are discovering sustainable alternative approaches to pesticides and chemical fertilizers.
Natural resources are decreasing with the passage of time due to spontaneously increasing world population.Hence, it is dire need of the hour to find out an alternative and viable approach for growing more food in such a manner that can reduce detrimental environmental impacts of intensive farming, fungicide resistance and environmental pollution.Among these approaches, biological control of pathogens by using endophytes is a cost effective and environment safe approach to minimize the use of pesticides in cereals.
Endophytes are metabolically active fungal or bacterial microbes with internal colonization, inconspicuous infections, transiently symptomless and diverse life history strategies [7][8].The beneficial impact of endophytes to plant diseases have amplified the concern of farmers and researchers for augmenting agricultural production.Endophytes antagonise disease pathogens directly by hyperparasitism, production of lytic enzymes and antimicrobial metabolites, indirectly by inducing systemic resistance, contending for space and nutrients and endorsing plant growth [9][10].In our previous research, numerous fungi that colonized different desert plants endophytically have been screened to evaluate their potential to promote wheat growth and to combat leaf rust pathogens.Four of the selected fungal endophytes displayed great potential in controlling Puccinia recondita causing wheat leaf rust in field conditions [11].In present study, we screened several fungal endophytes and investigated the inhibitory capacities of the four selected isolates against Puccinia striiformis to wheat susceptible genotypes in artificially inoculated diseased conditions.

Experimental Site and Sowing Conditions
Fifty local genotypes of wheat were sown for screening to yellow rust in the reseachh field area of Bahauddin Zakariya University, Multan during December, 2015.Each genotype was planted in experimental plot size of 1.2 m x 2.5 m surrounded by three rows of highly susceptible genotype Morocco.Rust inoculation methods like needle injection, rubbing, spraying and dusting with talcum powder were exploited at tillering and heading stage on Morocco for the development of a heavy pressure of rust infection [11][12]

Data Recording of Yellow Rust
Rust severity in percentage and host response was recorded by modified Cobb's scale described by Peterson [13].Rust severity was recorded four times with the consecutive gap period of 10 days while Morocco expressed 40-50% severity.Rating of the final disease severity (FDS) was noted as Morocco showed 90-100% severity.Coefficient of infection (CI) values were calculated for the rating of host responses of susceptible (S), resistant (R), moderately susceptible (MS) and moderately resistant (MR) [14].For each genotype, area under disease progress curve (AUDPC) with 10 days interval was calculated by the following equation [15].

Isolation of Fungal Endophytes
Samples of naturally occurring healthy roots, leaves and stems were taken at random from the various desert locations of Thar, Cholistan and Rohi and were shifted to the mycology and genetics lab through ice bucket for isolation of endophytes.Samples were sterilized, incubated for the emergence of endophytic fungi, purified and identified by the techniques described previously [11].

Optimization of efficient and compatible endophytic fungi with wheat seed
Spores of many endophytic fungi were harvested in distilled water by rubbing the surface of a sporulating pure culture with a sterile bent glass rod and maintained the spore suspension of 1×10 6 ml/L by dilution method.Germinating wheat seed were kept in test tubes containing 0.3% agar concentration in distilled water with fungal spore suspension of 1×10 6 ml/L and were incubated.After suitable intervals, root and shoot length of wheat seedlings were measured for investigating the efficacy of fungal endophytes.Consequently four best endophytes were selected for further experimentation.

In-Vivo Potential of Fungal Endophytes
Seeds of two certain yellow rust susceptible genotypes of wheat were soaked separately for 24 h in spore suspensions of four nominated (from lab experiments) efficient and compatible endophytes.During last week of November, 2015, these seeds were sown under randomized complete block design through factorial arrangement repeated thrice and untreated as control.
Inoculation was done artificially as performed in screening experiment.The FDS (%), AUDPC value, 1000-grain weight (g), Grain yield (gm -2 ) and yield increased (%) were measured for assessing the potential of fungal endophytes against yellow rust fungus P.
striiformis as well as their symbiotic response for rust susceptible genotypes in disease vulnerable conditions.The endophytes were re-isolated and identified from the inoculated host plants to confirm the colonization of the fungal endophytes within tissues.

Statistical analysis
Data were analysed using analysis of variance (ANOVA) and Dunkun's New Multiple Range Test (DNMRT), Tukey's test at 5% probability level in screening experiment and Least Significant Difference (LSD) test for other experiment [16]

Yellow Rust Susceptibility of Wheat Genotypes
The yellow rust significantly affected the local wheat genotypes presenting final disease severity ranged from 20-80% (

DISCUSSION
Although a number of the reports endorse the improvement in wheat growth by the exogenous employment of P. indica and substantiated that its association with host augmented the defence mechanisms, conferred disease tolerance and amplified wheat productivity and yield [17][18] but none is available in prior findings where P. indica and other fungal endophytes antagonised wheat yellow rust in field.In present study, fungal endophytes were utilized for the improvement of the wheat growth and yield in yellow rust disease conditions.Exploitation of fungal endophytes ascertained extremely efficacious in enriching the yield of susceptible genotypes (Fareed-06 and Shafaq-06).P. indica exhibited In our previous studies [11] paramount valuable outcomes followed by T. viride, C. lindemuthianum and A. lolii for prompting tolerance against P. striiformis.Findings of current study validated better grains weight and yield interconnected principally to the decreased disease severity of the endophytic associated wheat host.Enlarged photosynthetic area and net assimilation efficiency of host inferred the potentially antagonistic and aggressive role of fungal endophytes.Decreased disease severity details of larger surface area for producing and partitioning of photoassimilates towards reproductive growth, thus, better grains weight and yield.The potential of fungal endophytes against yellow rust fungus P. striiformis contributed substantially for stabilizing susceptible wheat host in disease conditions by stimulating disease tolerance.Alike, Rodriguez and Suryanarayanan[19][20] informed antagonistic role of C. lindemuthianum in Solanum lycopersicum with augmented disease resistance and improved growth and biomass as witness in current study.Numerous studies conveyed the antagonistic role of T. viride as well as other Trichoderma spp.for prompting positive impact to the hosts and copping with diverse diseases[21][22][23].

Table 1
entitled as susceptible, whereas both Faisalabad-08 and Bhakhar-08 depicted the CI values of 8.0, and also the lowest AUDPC values of 200 and 265, thus, were highly resistant to the yellow rust pathogen (Puccinia striiformis).The rest of the genotypes were ranked as moderately resistant to susceptible depicting the range of values 300 to 465 for portraying their resistance as well as 475 to 850 susceptibility (Table1).

Table 2
Puccinia striiformis were contributed significantly for alleviating wheat plant to yellow rust through antagonising the disease attack by subsiding disease severity consequently enhanced thousand grain weight and grain yield (Table2).P. indica indicated significant performance by increasing 12.3% grain yield comparing to control followed by T. viride 10.6 %, C. lindemuthianum 6.2% and A. lolii 4.2% in yellow rust conditions.Both grains weight and yield were effectually improved by the symbiosis of T. viride and P. indica while the C. lindemuthianum and A. lolii revealed moderate performances for conferring tolerance against Puccinia striiformis.
Symbiotic impact of fungal endophytes to susceptible wheat genotypes and potentially aggressive input for

Table 2 : Potential of fungal endophytes confronting fungus of wheat yellow rust P. striiformis in field conditions FDS=
, the four fungal endophytes P. indica, T. viride, C. lindemuthianum and A. lolii showed paramount valuable outcomes for prompting tolerance against fungus P. recondita in leaf rust susceptible wheat genotypes by decreasing final disease severity (FDS) 30-60% and area under disease progress curve (AUDPC), resultantly achieved the 12.2% 10.6%, 06.2% and 04.2% grain yield gain, respectively.The outcomes of current study implied that symbiotic association of T. viride, P. indica, A. lolii and C. lindemuthianum with wheat host could induce tolerance against yellow rust.FDS= Final disease severity, AUDPS= Area under disease progress curve, CI= Coefficient of infection, IR= Infection response Final disease severity, AUDPS= Area under disease progress curve, TGW= Thousand grains weight, GY= Grain yield, YI= Yield increased CONCLUSIONSFungal endophytes can protect Triticum aestivum (bread wheat) from destruction caused by fungus P. striiformis through declining the disease severity and subsequently improve the grain yield in field conditions.