Molecular characterization of a novel mycovirus isolated from Rhizoctonia solani AG-1 IA strain 9-11.

The complete genome sequence of a double-stranded RNA (dsRNA) virus, Rhizoctonia solani dsRNA virus 11 (RsRV11), isolated from Rhizoctonia solani AG-1 IA strain 9-11 was determined. The RsRV11 genome is 9,555 bp in length and contains three conserved domains: structural maintenance of chromosomes (SMC) superfamily, phosphoribulokinase (PRK), and RNA-dependent RNA polymerase (RdRp). The RsRV11 genome has two non-overlapping open reading frames (ORFs). ORF1 is predicted to encode a 204.12-kDa protein that shares low but significant amino acid sequence similarity with a putative protein encoded by Rhizoctonia solani RNA virus HN008 (RsRV-HN008). ORF2 potentially encodes a 132.41-kDa protein that contains the conserved domain of the RdRp. Phylogenetic analysis indicated that RsRV11 clustered with RsRV-HN008 in a separate clade from other virus families. This implies that RsRV11 and RsRV-HN008 should be included in a new mycovirus taxon close to the family Megabirnaviridae and that RsRV11 is a new mycovirus.

DNA barcode, multiplex PCR and qPCR assay for diagnosis of pathogens infecting pulse crops to facilitate safe exchange and healthy conservation of germplasm.

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > ß-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components-plant, pathogen, and environment-involved in the disease triangle. Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

A rapid colorimetric LAMP assay for detection of Rhizoctonia solani AG-1 IA causing sheath blight of rice.

Rhizoctonia solani is one of the most devastating pathogens. R. solani AG-1 IA causes sheath blight in rice, maize, and other Gramineous plants. Accurate identification is essential for the effective management of this pathogen. In the present study, a set of four primers were designed viz. RSPG1, RSPG2, RSPG4, and RSPG5 for polygalacturonase (PG) gene, an important virulence factor in phytopathogenic fungi. All four primer sets showed specific amplification of 300 bp (RSPG1F/R), 375 bp (RSPG2F/R), 500 bp (RSPG4F/R) and 336 bp (RSPG5F/R) amplicons. q-PCR detection using each primer sets could detect up to 10 pg of DNA. We also designed six primers (RS_pg_F3_1/RS_pg_B3_1, RS_pg_FIP_1.1/RS-pg_BIP_1.1, and RS_pg_LF_1/RS_pg_LB_1) for PG gene. Further, a colorimetric LAMP assay developed yielded visual confirmation of the pathogen within 45 min of sample collection when coupled with rapid high throughput template preparation method (rHTTP) from infected samples. The sensitivity of the LAMP assay was as low as 1.65 fg/µl of template DNA and could effectively detect R. solani AG-1 IA from diseased plant tissues and soil samples. The LAMP assay was highly specific for R. solani as it did not show any amplification with other AG groups of R. solani and closely related fungal and bacterial outgroups. This study will help in designing an effective point of care diagnostic method for early monitoring of R. solani and thereby planning timely preventive measures against the pathogen.

A longitudinal study on morpho-genetic diversity of pathogenic Rhizoctonia solani from sugar beet and dry beans of western Nebraska.

BACKGROUND: Root and stem rot caused by Rhizoctonia solani is a serious fungal disease of sugar beet and dry bean production in Nebraska. Rhizoctonia root rot and crown rot in sugar beet and dry bean have reduced the yield significantly and has also created problems in storage. The objective of this study was to analyze morpho-genetic diversity of 38 Rhizoctonia solani isolates from sugar beet and dry bean fields in western Nebraska collected over 10 years. Morphological features and ISSR-based DNA markers were used to study the morphogenetic diversity. RESULTS: Fungal colonies were morphologically diverse in shapes, aerial hyphae formation, colony, and sclerotia color. Marker analysis using 19 polymorphic ISSR markers showed polymorphic bands ranged from 15 to 28 with molecular weight of 100 bp to 3 kb. Polymorphic loci ranged from 43.26-92.88%. Nei genetic distance within the population ranged from 0.03-0.09 and Shannon diversity index varied from 0.24-0.28. AMOVA analysis based on ΦPT values showed 87% variation within and 13% among the population with statistical significance (p < 0.05). Majority of the isolates from sugar beet showed nearby association within the population. A significant number of isolates showed similarity with isolates of both the crops suggesting their broad pathogenicity. Isolates were grouped into three different clusters in UPGMA based cluster analysis using marker information. Interestingly, there was no geographical correlation among the isolates. Principal component analysis showed randomized distribution of isolates from the same geographical origin. Identities of the isolates were confirmed by both ITS-rDNA sequences and pathogenicity tests. CONCLUSION: Identification and categorization of the pathogen will be helpful in designing integrated disease management guidelines for sugar beet and dry beans of mid western America.

Synthesis and Biological Evaluation of Novel Benodanil-Heterocyclic Carboxamide Hybrids as a Potential Succinate Dehydrogenase Inhibitors.

In order to discover new antifungal agents, twenty novel benodanil-heterocyclic carboxamide hybrids were designed, synthesized, and characterized by 1H NMR and HRMS. In vitro, their antifungal activities against four phytopathogenic fungi were evaluated, as well as some of the target compounds at 50 mg/L demonstrated significant antifungal activities against Rhizoctonia solani. Especially, compounds 17 (EC50 = 6.32 mg/L) and 18 (EC50 = 6.06 mg/L) exhibited good antifungal activities against R. solani and were superior to the lead fungicide benodanil (a succinate dehydrogenase inhibitor, SDHI) (EC50 = 6.38 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media with the addition of compound 17 grew abnormally as compared with the negative control with tenuous, wizened, and overlapping colonies, and compounds 17 (IC50 = 52.58 mg/L) and 18 (IC50 = 56.86 mg/L) showed better inhibition abilities against succinate dehydrogenase (SDH) than benodanil (IC50 = 62.02 mg/L). Molecular docking revealed that compound 17 fit in the gap composed of subunit B, C, and D of SDH. Furthermore, it was shown that the main interaction, one hydrogen bond interaction, was observed between compound 17 and the residue C/Trp-73. These studies suggested that compound 17 could act as a potential fungicide to be used for further optimization.

Real-time PCR quantification of Rhizoctonia solani AG-3 from soil samples.

Rhizoctonia solani anastomosis group 3 (AG-3) causes several diseases of potato, including black scurf and stem canker, affecting potato production in the Skagit Valley, Washington, and around the world. Primers for a SYBR-Green II-based real-time polymerase chain reaction (qPCR) assay were designed from sequences of the nuclear internal transcribed spacer (ITS) regions of fungal isolates of potato and onion from the Pacific Northwest, USA. The primers preferentially amplified R. solani AG-3 DNA, compared to DNA from R. solani AG-4, AG-5 and AG-8. In silico analysis of primer-template duplex stability indicated that the assay also will detect R. solani AG-3 isolates from pea and onion in Washington State and from diverse crop species around the world, but not R. solani AG-9 and AG-2-1. The assay was used to quantify R. solani AG-3 populations in pathogen-infested field soils after temporary flooding rotation, a practice found to be effective for reducing Sclerotinia sclerotiorum and R. solani AG-3 in potatoes in growth chamber studies. Population densities of the pathogen were not significantly reduced in saturated (flooded) soils relative to fallow. However, the qPCR approach was more sensitive and quantitative than the toothpick baiting method for diagnosis of these soil samples. Accurate detection and quantification of R. solani AG-3 in soil will facilitate the development of integrated management plans for Rhizoctonia diseases of potato.

Anastomosis Groups of Rhizoctonia solani associated with tomato foot rot in Pothohar Region of Pakistan.

Rhizoctonia solani Kühn (teleomorph = Thanatephorus cucumeris (Frank) Donk) is one of the important soil-borne fungal pathogen, which infects tomato with typical symptoms of seedling damping-off and foot rot. During surveys (2014 and 2015 crop season) of nine tomato growing areas in Pothohar region of Pakistan, symptoms of foot rot were noted on approximately 33.4% of the plants observed at soil line level of the stem. Lesions on infected plant stems were irregular in shape, water-soaked, brown in colour manifesting sunken appearance. Fungal colonies isolated from stem portions of the diseased plants on malt extract agar medium were light grey to brown in colour with abundant mycelial growth and branched hyphae. A septum was always present in the branch of hyphae near the originating point with a slight constriction at the branch. No conidia or conidiophores were observed. All isolates were multinucleate when subjected to DAPI (4',6-diamidino-2-phenylindole) stain. Based on morphological characteristics of fungal hyphae, isolates were identified as R. solani. Restriction analysis of PCR-amplified ribosomal DNA with four discriminant enzymes (MseI, AvaII, HincII, and MunI) and hyphal interactions with known tester strains confirmed these isolates belong to AG-3-PT (64.2%), AG-2-1 (14.2%), AG-2-2 (9.5%), AG-5 (7.1%) and AG-4-HGI (4.7%). AG-3-PT was widely distributed to major tomato growing areas while other groups were confined to distinct locations. Internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced which had 99-100% identity with the corresponding gene sequences of respective R. solani AGs. To confirm Koch's postulates, four week old tomato plants were transplanted into 1.5 L plastic pots containing sterilized potting mixture i.e. sand: clay: farmyard manure, at the rate of 1:1:1. Soil inoculum containing 10 g of barley grains colonized with each isolate of R. solani for 14 days was mixed in the upper 2 cm layer of soil (Taheri and Tarighi, 2012). A set of uninoculated plants was used as a control. Ambient conditions were provided under the greenhouse. 21 days after inoculation, water-soaked greyish to brown lesions similar to the symptoms of the previous infection were observed on stem portions of all inoculated plants while control plants remained symptomless. Fungus re-isolated from infections was confirmed as R. solani by microscopic appearance of the hyphae. Present study is the first report of AG composition of R. solani infecting tomato in Pakistan which will be useful to breeding programs working on varietal evaluation.

Differentiation of mixed soil-borne fungi in the genus level using infrared spectroscopy and multivariate analysis.

Early detection of soil-borne pathogens, which have a negative effect on almost all agricultural crops, is crucial for effective targeting with the most suitable antifungal agents and thus preventing and/or reducing their severity. They are responsible for severe diseases in various plants, leading in many cases to substantial economic losses. In this study, infrared (IR) spectroscopic method, which is known as sensitive, accurate and rapid, was used to discriminate between different fungi in a mixture was evaluated. Mixed and pure samples of Colletotrichum, Verticillium, Rhizoctonia, and Fusarium genera were measured using IR microscopy. Our spectral results showed that the best differentiation between pure and mixed fungi was obtained in the 675-1800 cm-1 wavenumber region. Principal components analysis (PCA), followed by linear discriminant analysis (LDA) as a linear classifier, was performed on the spectra of the measured classes. Our results showed that it is possible to differentiate between mixed-calculated categories of phytopathogens with high success rates (~100%) when the mixing percentage range is narrow (40-60) in the genus level; when the mixing percentage range is wide (10-90), the success rate exceeded 85%. Also, in the measured mixed categories of phytopathogens it is possible to differentiate between the different categories with ~100% success rate.

UPLC-QTOF-MS metabolomics analysis revealed the contributions of metabolites to the pathogenesis of Rhizoctonia solani strain AG-1-IA.

To explore the pathogenesis of Rhizoctonia solani and its phytotoxin phenylacetic acid (PAA) on maize leaves and sheaths, treated leaf and sheath tissues were analyzed and interpreted by ultra-performance liquid chromatography-mass spectrometry combined with chemometrics. The PAA treatment had similar effects to those of R. solani on maize leaves regarding the metabolism of traumatin, phytosphingosine, vitexin 2'' O-beta-D-glucoside, rutin and DIBOA-glucoside, which were up-regulated, while the synthesis of OPC-8:0 and 12-OPDA, precursors for the synthesis of jasmonic acid, a plant defense signaling molecule, was down-regulated under both treatments. However, there were also discrepancies in the influences exhibited by R. solani and PAA as the metabolic concentration of zeaxanthin diglucoside in the R. solani infected leaf group decreased. Conversely, in the PAA-treated leaf group, the synthesis of zeaxanthin diglucoside was enhanced. Moreover, although the synthesis of 12 metabolites were suppressed in both the R. solani- and PAA-treated leaf tissues, the inhibitory effect of R. solani was stronger than that of PAA. An increased expression of quercitrin and quercetin 3-O-glucoside was observed in maize sheaths treated by R. solani, while their concentrations were not changed significantly in the PAA-treated sheaths. Furthermore, a significant decrease in the concentration of L-Glutamate, which plays important roles in plant resistance to necrotrophic pathogens, only occurred in the R. solani-treated sheath tissues. The differentiated metabolite levels may be the partial reason of why maize sheaths were more susceptible to R. solani than leaves and may explain the underlying mechanisms of R. solani pathogenesis.

Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon.

Rhizoctonia solani is a soil-borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)-dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone-induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA-deficient NahG transgenic rice and the sheath blight-resistant B. distachyon accessions, Bd3-1 and Gaz-4, which activate SA-dependent signaling on inoculation. Our findings suggest a hemi-biotrophic nature of R. solani, which can be targeted by SA-dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

Seasonal dynamics and fungicide sensitivity of organisms causing brown patch of tall fescue in North Carolina.

Brown patch, caused by multiple species of Rhizoctonia and Rhizoctonia-like fungi, is the most severe summer disease of tall fescue in home lawns across the southeastern United States. Home lawns were surveyed in central North Carolina from 2013 to 2015 to determine the organisms present during typical epidemics of brown patch in tall fescue. Isolates of Rhizoctonia and Rhizoctonia-like fungi were obtained by sampling 147 locations in July 2013 and May and July 2014. In addition, 11 sites were sampled once a week for 12 consecutive weeks from late May to the end of July 2015. All isolates were identified to species and anastomosis group with nuc rDNA internal transcribed spacer (ITS) sequence analysis. Isolations from brown patch lesions in May 2014 predominately yielded Ceratobasidium cereale (77% of the organisms recovered), whereas the organisms recovered in July 2013 and 2014 were R. solani AG 2-2-IIIB (44%), R. solani AG 1-IB (37%), and R. zeae (14%). In 2015, Ceratobasidium cereale was isolated from all 11 locations in May but was replaced by Rhizoctonia species in June and July. Sensitivity of the May 2014 isolates to multiple concentrations of the fungicides azoxystrobin, flutolanil, fluxapyroxad, and propiconazole was compared with sensitivity of isolates collected in 2003, to determine whether multiple years of exposure to fungicides applied for brown patch control had altered fungicide sensitivity. Historical isolates of R. solani, which had never been exposed to fungicide applications for brown patch control, were also included for comparison. Mean EC50 values (concentration of fungicide needed to inhibit mycelial growth by 50%) varied across fungicides and species, but no resistance was observed, and there was no apparent shift in sensitivity over the years. An additional 94 isolates from 2015 were screened against azoxystrobin, flutolanil, fluxapyroxad, and propiconazole, and fungicide insensitivity was not observed.

Nutrients and host attributes modulate the abundance and functional traits of phyllosphere microbiome in rice.

The abundance of phyllosphere bacterial communities of seven genotypes of rice ADT- 38, ADT-43, CR-1009, PB-1, PS-5, P-44, and PB-1509 was investigated, in relation to nutrient dynamics of rhizosphere and leaves. P-44 genotype recorded highest pigment accumulation, while genotypes CR-1009 and P-44 exhibited most number of different bacterial morphotypes, Colony forming units in two media (Nutrient agar and R2A) varied significantly and ranged from 106-107 per g plant tissues. Among the selected 60 distinct morphotypes, IAA and siderophore producers were the dominant functional types. Biocontrol activity against Drechslera oryzae was shown by 38 isolates, while 17 and 9 isolates were potent against Rhizoctonia solani and Magnaporthe oryzae respectively. Principal Component Analysis (PCA) illustrated the significant effects of selected soil and leaf nutrients of seven rice varieties on the culturable phyllospheric population (log CFU), particularly in the R2A medium. Eigen values revealed that 83% of the variance observed could be assigned to Leaf-Fe, Leaf-Mn, chlorophyll b and soil organic carbon (OC). Quantitative PCR analyses of abundance of bacteria, cyanobacteria and archaebacteria revealed a host-specific response, with CR-1009 showing highest number of 16S rRNA copies of bacterial members, while both P-44 and PS-5 had higher cyanobacterial abundance, but lowest number of those belonging to archaebacteria. Nutritional aspects of leaf and soil influenced the abundance of bacteria and their functional attributes; this is of interest for enhancing the efficacy of foliar inoculants, thereby, improving plant growth and disease tolerance.

Comparative secretome analysis of Rhizoctonia solani isolates with different host ranges reveals unique secretomes and cell death inducing effectors.

Rhizoctonia solani is a fungal pathogen causing substantial damage to many of the worlds' largest food crops including wheat, rice, maize and soybean. Despite impacting global food security, little is known about the pathogenicity mechanisms employed by R. solani. To enable prediction of effectors possessing either broad efficacy or host specificity, a combined secretome was constructed from a monocot specific isolate, a dicot specific isolate and broad host range isolate infecting both monocot and dicot hosts. Secretome analysis suggested R. solani employs largely different virulence mechanisms to well-studied pathogens, despite in many instances infecting the same host plants. Furthermore, the secretome of the broad host range AG8 isolate may be shaped by maintaining functions for saprophytic life stages while minimising opportunities for host plant recognition. Analysis of possible co-evolution with host plants and in-planta up-regulation in particular, aided identification of effectors including xylanase and inhibitor I9 domain containing proteins able to induce cell death in-planta. The inhibitor I9 domain was more abundant in the secretomes of a wide range of necrotising fungi relative to biotrophs. These findings provide novel targets for further dissection of the virulence mechanisms and potential avenues to control this under-characterised but important pathogen.

An improved strategy for stable biocontrol agents selecting to control rice sheath blight caused by Rhizoctonia solani.

Rice sheath blight caused by Rhizoctonia solani Kühnis increasingly threatening rice production in China. DNA fingerprints of 220 R. solani strains isolated in 11 provinces of China were established by random amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of strains isolated from the same region showed high similarity, indicating that the genetic diversity of R. solani strains is significantly related to geographical origin. We assessed potential bio-control abilities of bio-control agents (BCAs) by values according to inhibition zones against R. solani, extracellular hydrolytic enzymes activity and siderophores production in vitro. Fourteen strains with diverse expected bio-control potential were tested for their bio-control efficacy against rice sheath blight caused by 11 pathogenic exemplars and for growth promoting ability, separately. Bio-control efficacy of single bacterium against various R. solani strains differed significantly (-36.23%∼88.24%), while Pseudomonas fluorescens 4aYN11 achieved a relatively stable control efficacy of 32.26%-78.79% and growth promotion of 18.43%. Pearson correlation coefficient between bio-control efficacy of each BCAs and their assessment is 0.717. In the present study, we established an improved strategy for screening stable bio-control agents based on an assessment system, their growth promotion potential and phylogenetic diversity of pathogen R. solani, and the result provides us not only one promising bio-control strain 4aYN11 with an average bio-control efficacy of 56.50%, but also a practical way for future screen of novel BCAs.

Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3.

The basidiomycetes fungus Rhizoctonia solani AG3 is responsible for black scurf disease on potato and occurs in each potato growing area world-wide. In this study, the draft genome sequence of the black scurf pathogen R. solani AG3-PT isolate Ben3 is presented. The genome sequence of R. solani AG3-PT isolate Ben3 consists of 1385 scaffolds. These scaffolds amount to a size of approx. 51 Mb. Considering coverage analyses of contigs, the size of the diploid genome was estimated to correspond to 116 Mb. Gene prediction by applying AUGUSTUS (3.2.1.) resulted in 12,567 identified genes. Based on automatic annotation using GenDBE, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the R. solani AG3-PT isolate Ben3 genome. Comparative analyses including the R. solani AG3 isolate Rhs1AP, also originating from potato, revealed first insights into core genes shared by both isolates and unique determinants of each isolate.

Variation of rDNA Internal Transcribed Spacer Sequences in Rhizoctonia cerealis.

Fifty-four single protoplast isolates (SPIs) were regenerated from three Rhizoctonia cerealis strains. A total of 169 rDNA-ITS regions were cloned and sequenced from these 54 SPIs. Variations in the ITS1 and ITS2 regions that flank the 5.8S gene were found within clones from the same strain, as well as within clones from the same SPI. These include variations in GC content and ITS length, and single-nucleotide polymorphisms (SNPs). The different strains and SPIs GC contents range from 40.25 to 41.74% and from 42.40 to 45.02%, in the ITS1 and ITS2 regions, respectively. All SNPs occur in the ITS1 and ITS2 regions, with 3-6 and 4-6 polymorphic sites in each region, respectively, in the different strains. SNP variation is relatively stable within the same strain. For example, the 89 ITS sequences generated from isolate WK-207, regardless of SPI or clone, predominantly cluster into two separate clades on a phylogenetic tree, suggesting that nuclei genetic heterogeneity is related to ITS variation in R. cerealis. Although rDNA-ITS sequences from the three strains and different SPIs are somewhat variable, all of our ITS sequences cluster together in anastomosis subgroup AG-DI during phylogenetic analysis. The ITS variation we observed does not negatively influence R. cerealis anastomosis group or subgroup classification.

Rapid and sensitive diagnoses of dry root rot pathogen of chickpea (Rhizoctonia bataticola (Taub.) Butler) using loop-mediated isothermal amplification assay.

Dry root rot (DRR) caused by the fungus Rhizoctonia bataticola (Taub.) Butler, is an emerging disease in chickpea. The disease is often mistaken with other root rots like Fusarium wilt, collar rot and black root rot in chickpea. Therefore, its timely and specific detection is important. Current detection protocols are either based on mycological methods or on protocols involving DNA amplification by polymerase chain reaction (PCR). Here we report the rapid and specific detection of R. bataticola using loop-mediated isothermal amplification (LAMP) assay targeting fungal specific 5.8S rDNA sequence for visual detection of R. bataticola. The reaction was optimized at 63 °C for 75 min using minimum 10 fg of DNA. After adding SYBR Green I in LAMP products, the amplification was found to be highly specific in all the 94 isolates of R. bataticola collected from diverse geographical regions as well as DRR infected plants and sick soil. No reaction was found in other pathogenic fungi infecting chickpea (Fusarium oxysporum f. sp. ciceris, Rhizoctonia solani, Sclerotium rolfsii and Fusarium solani) and pigeonpea (Fusarium udum and Phytophthora cajani). The standardised LAMP assay with its simplicity, rapidity and specificity is very useful for the visual detection of this emerging disease in chickpea.