Spatial distribution and identification of potential risk regions to rice blast disease in different rice ecosystems of Karnataka.

Rice is a globally important crop and highly vulnerable to rice blast disease (RBD). We studied the spatial distribution of RBD by considering the 2-year exploratory data from 120 sampling sites over varied rice ecosystems of Karnataka, India. Point pattern and surface interpolation analyses were performed to identify the spatial distribution of RBD. The spatial clusters of RBD were generated by spatial autocorrelation and Ripley's K function. Further, inverse distance weighting (IDW), ordinary kriging (OK), and indicator kriging (IK) approaches were utilized to generate spatial maps by predicting the values at unvisited locations using neighboring observations. Hierarchical cluster analysis using the average linkage method identified two main clusters of RBD severity. From the Local Moran's I, most of the districts were clustered together (at I > 0), except the coastal and interior districts (at I < 0). Positive spatial dependency was observed in the Coastal, Hilly, Bhadra, and Upper Krishna Project ecosystems (p > 0.05), while Tungabhadra and Kaveri ecosystem districts were clustered together at p < 0.05. From the kriging, Hilly ecosystem, middle and southern parts of Karnataka were found vulnerable to RBD. This is the first intensive study in India on understanding the spatial distribution of RBD using geostatistical approaches, and the findings from this study help in setting up ecosystem-specific management strategies against RBD.

Host range and virulence diversity of Pectobacterium carotovorum subsp. brasiliense strain RDKLR infecting radish in India, and development of a LAMP-based diagnostics.

AIM: This work aimed at determining the pathogenicity, molecular characterization, host range and rapid detection of Pectobacterium carotovorum subsp. brasiliense (Pcb) causing soft rot disease in radish. METHODS AND RESULTS: The four isolated isolates were inoculated to radish, typical soft rot symptoms were observed and Koch's postulates were proved. The most virulent strain RDKLR was morphologically and biochemically distinct. Pcb showed a positive potato soft rot test and elicited hypersensitivity response on Nicotiana tobaccum. The genes Pel2 and pmrA were used for subspecies characterization of Pcb. It has a wide host range and infection was observed on slices of carrot, tomato, radish, potato, cauliflower, cabbage, chilli, knol-khol, bell pepper and cucumber. Infectivity was also seen in seedlings under glasshouse conditions. Pcb produced cell wall degrading enzymes in semi-quantification assay and is a strong biofilm producer. The LAMP technique was standardized to help rapid detection and take prophylactic measures to manage the disease. CONCLUSION: This work reports Pcb as a new soft rot causing organism of radish in India. Pcb is highly virulent with a broad host range. The LAMP technique helps in rapid detection. SIGNIFICANCE AND IMPACT OF THE STUDY: Pcb-induced soft rot causes significant yield loss, decreased market value, damage in transit, storage and the market. Disease characterization and early identification aid in disease management and prevention in the field.

Multilocus sequence analysis and identification of mating-type idiomorphs distribution in Magnaporthe oryzae population of Karnataka state of India.

AIMS: To investigate the genetic diversity, population structure and mating-type distribution among the eco-distinct isolates of Magnaporthe oryzae from Karnataka, India. METHODS AND RESULTS: A set of 38 isolates of M. oryzae associated with leaf blast disease of rice were collected from different rice ecosystems of Karnataka, India, and analysed for their diversity at actin, ß-tubulin, calmodulin, translation elongation factor 1-α (TEF-1-α), and internal transcribed spacer (ITS) genes/region. The isolates were grouped into two clusters based on the multilocus sequence diversity, the majority being in cluster-IA (n = 37), and only one isolate formed cluster-IB. Population structure was analysed using 123 SNP data to understand the genetic relationship. Based on K = 2 and ancestry threshold of >70%, blast strains were classified into two subgroups (SG1 and SG2) whereas, based on K = 4 and ancestry threshold of >70%, blast strains were classified into four subgroups (SG1, SG2, SG3 and SG4). We have identified 13 haplotype groups where haplotype group 2 was predominant (n = 20) in the population. The Tajima's and Fu's Fs neutrality tests exhibited many rare alleles. Further, the mating-type analysis was also performed using MAT1 gene-specific primers to find the potentiality of sexual reproduction in different ecosystems. The majority of the isolates (54.5%) had MAT1-2 idiomorph, whereas 45.5% of the isolates possessed MAT1-1 idiomorph. CONCLUSIONS: The present study found the genetically homogenous population of M. oryzae by multilocus sequence analysis. Both mating types, MAT1-1 and MAT1-2, were found within the M. oryzae population of Karnataka. SIGNIFICANCE AND IMPACT OF STUDY: The study on the population structure and sexual mating behaviour of M. oryzae is important in developing region-specific blast-resistant rice cultivars. This is the first report of MAT1 idiomorphs distribution in the M. oryzae population in any Southern state of India.

Morpho-molecular diversity and avirulence genes distribution among the diverse isolates of Magnaporthe oryzae from Southern India.

AIMS: To investigate the diversity of eco-distinct isolates of Magnaporthe oryzae for their morphological, virulence and molecular diversity and relative distribution of five Avr genes. METHODS AND RESULTS: Fifty-two M. oryzae isolates were collected from different rice ecosystems of southern India. A majority of them (n = 28) formed a circular colony on culture media. Based on the disease reaction on susceptible cultivar (cv. HR-12), all 52 isolates were classified in to highly virulent (n = 28), moderately virulent (n = 11) and less-virulent (13) types. Among the 52 isolates, 38 were selected for deducing internal transcribed spacer (ITS) sequence diversity. For deducing phylogeny, another set of 36 isolates from other parts of the world was included, which yielded two distinct phylogenetic clusters. We identified eight haplotype groups and 91 variable sites within the ITS sequences, and haplotype-group-2 (Hap_2) was predominant (n = 24). The Tajima's and Fu's Fs neutrality tests exhibited many rare alleles. Furthermore, PCR analysis for detecting the presence of five Avr genes in the different M. oryzae isolates using Avr gene-specific primers in PCR revealed that Avr-Piz-t, Avr-Pik, Avr-Pia and Avr-Pita were present in 73.68%, 73.68%, 63.16% and 47.37% of the isolates studied, respectively; whereas, Avr-Pii was identified only in 13.16% of the isolates. CONCLUSIONS: Morpho-molecular and virulence studies revealed the significant diversity among eco-distinct isolates. PCR detection of Avr genes among the M. oryzae population revealed the presence of five Avr genes. Among them, Avr-Piz-t, Avr-Pik and Avr-Pia were more predominant. SIGNIFICANCE AND IMPACT OF THE STUDY: The study documented the morphological and genetic variability of eco-distinct M. oryzae isolates. This is the first study demonstrating the distribution of the Avr genes among the eco-distinct population of M. oryzae from southern India. The information generated will help plant breeders to select appropriate resistant gene/s combinations to develop blast disease-resistant rice cultivars.

Comparative metagenomic analysis of rice soil samples revealed the diverse microbial population and biocontrol organisms against plant pathogenic fungus Magnaporthe oryzae.

Intensive cropping degrades soil quality and disrupts the soil microbiome. To understand the effect of rice monocropping on soil-microbiome, we used a comparative 16S rRNA metagenome sequencing method to analyze the diversity of soil microflora at the genomic level. Soil samples were obtained from five locations viz., Chamarajnagara, Davangere, Gangavathi, Mandya, and Hassan of Karnataka, India. Chemical analysis of soil samples from these locations revealed significant variations in pH (6.00-8.38), electrical conductivity (0.17-0.69 dS m-1), organic carbon (0.51-1.29%), available nitrogen (279-551 kg ha-1), phosphorous (57-715 kg ha-1) and available potassium (121-564 kg ha-1). The 16S metagenome analysis revealed that the microbial diversity in Gangavathi soil samples was lower than in other locations. The soil sample of Gangavathi showed a higher abundance of Proteobacteria (85.78%) than Mandya (27.18%). The Firmicutes were more abundant in Chamarajnagar samples (36.01%). Furthermore, the KEGG pathway study revealed enriched nitrogen, phosphorus, and potassium metabolism pathways in all soil samples. In terms of the distribution of beneficial microflora, the decomposers were more predominant than the nutrient recyclers such as nitrogen fixers, phosphorous mineralizers, and nitrifiers. Furthermore, we isolated culturable soil microbes and tested their antagonistic activity in vitro against a fungal pathogen of rice, Magnaporthe oryzae strain MG01. Six Bacillus sp. and two strains of Trichoderma harzianum showed higher antagonistic activity against MG01. Our findings indicate that metagenome sequencing can be used to investigate the diversity, distribution, and abundance of soil microflora in rice-growing areas. The knowledge gathered can be used to develop strategies for maintaining soil quality and crop conservation to increase crop productivity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02783-y.

First report of Alternaria alternata causing leaf spot on Oat (Avena sativa) in India.

Oat (Avena sativa L.) is an important cereal crop grown worldwide primarily for food and animal feed. In November 2019, a leaf spot disease was observed on the oat plants at Mandya (12.5218° N, 76.8951° E), Karnataka, India. The disease incidence on plants was ranged between 43 to 57 percent. Initially, the symptoms appeared on leaves as small dark-brown spots surrounded by a yellow halo later turned to irregular necrotic spots with a yellow halo. A total of thirty leaves showing typical symptoms were collected from ten plants (three leaves per plant), cut into an area of 4-5 mm pieces at the junction of infected and healthy tissues. Cut tissues were soaked in 75% ethanol for 45 seconds, followed by 1% sodium hypochlorite solution for 1 min, rinsed five times in sterile distilled water, air dried, and placed on PDA and incubated at 25 ± 1 ℃. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for five isolates. The colonies initially produced light-greyish aerial mycelia, then turned to dark-greyish upon maturity. Conidia were obclavate to pyriform and measured 17.34 to 46.97 µm long, 5.38 to 14.31 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2.73 to 10.17µm) (n = 50.) Based on the morpho-cultural characteristics, the isolates were identified as Alternaria spp., and PCR assay using species-specific primers (AAF2/AAR3; Konstantinova, et al. 2002) confirmed the taxonomic identity of all five isolates as A. alternata. To further confirm the identity, the internal transcribed spacer (ITS), small subunit (SSU), glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase second largest subunit (rpb2), Alternaria major allergen (Alt a1), endopolygalacturonase (endoPG), an anonymous gene region OPA10-2, KOG1058 and translation elongation factor 1-alpha (tef1) of two isolates MAAS-1 and MAAS-2 were PCR amplified using the primers described previously (Woudenberg et al. 2015; Praveen et al. 2020) and the resultant PCR products were sequenced and deposited in NCBI GenBank (ITS: MW487388, MW741962, SSU: MW506220, MW752854, gapdh: MW506221, MW752855, rpb2: MW506222, MW752856, Alt a1: MW506223, MW752857, endoPG: MW506224, MW752858, OPA10-2: MW506225, MW752859, KOG1058: MW506226, MW752860, and tef1: MW506227, MW752861) which showed (99.62%, 99.81%), (100%, 100%), (100%, 99.66%), (100%, 100%), (99.58%, 99.15%), (99.55%, 99.32%), (99.53%, 99.68%), (99.23%, 99.56%) and (99.17%, 99.58%) identity with ITS (AF347031), SSU (KC584507), gapdh (AY278808), rpb2 (KC584375), Alt a1 (AY563301), endoPG (JQ811978), OPA10-2 (KP124632), KOG1058 (KP125233) and tef1 (KC584634) genes/genomic regions of type strain CBS916.96 of A. alternata respectively, confirming the identity of MAAS-1 as A. alternata. For pathogenicity assay, the conidial suspension (2 × 106 conidia/ml) of MAAS-1 isolate was artificially sprayed until runoff on ten healthy oat plants (cv. Kent, 35 days old) and ten plants sprayed with sterile water served as control. All plants were covered with polyethylene covers and kept under the greenhouse at 28 ± 1 ℃. The pathogenicity assay was repeated three times. After six days post-inoculation, small dark-brown spots with light-yellow halo appeared on leaves of MAAS-1inoculated plants. In comparison, no symptoms were observed on control plants. The fungal pathogen was re-isolated from the artificially infected plants and confirmed as A. alternata based on morpho-cultural characteristics and PCR assays. The leaf spot disease of Oat caused by A. alternata has already been reported from China (Chen et al. 2020); to our knowledge, it is the first report of A. alternata causing leaf spot on Oat in India. The leaf spot disease is an emerging threat to oat cultivation, and it reduces the grain yield and leaf quality; therefore, its management is essential for increasing productivity.

LAMP-based foldable microdevice platform for the rapid detection of Magnaporthe oryzae and Sarocladium oryzae in rice seed.

Rice blast (caused by Magnaporthe oryzae) and sheath rot diseases (caused by Sarocladium oryzae) are the most predominant seed-borne pathogens of rice. The detection of both pathogens in rice seed is essential to avoid production losses. In the present study, a microdevice platform was designed, which works on the principles of loop-mediated isothermal amplification (LAMP) to detect M. oryzae and S. oryzae in rice seeds. Initially, a LAMP, polymerase chain reaction (PCR), quantitative PCR (qPCR), and helicase dependent amplification (HDA) assays were developed with primers, specifically targeting M. oryzae and S. oryzae genome. The LAMP assay was highly efficient and could detect the presence of M. oryzae and S. oryzae genome at a concentration down to 100 fg within 20 min at 60 °C. Further, the sensitivity of the LAMP, HDA, PCR, and qPCR assays were compared wherein; the LAMP assay was highly sensitive up to 100 fg of template DNA. Using the optimized LAMP assay conditions, a portable foldable microdevice platform was developed to detect M. oryzae and S. oryzae in rice seeds. The foldable microdevice assay was similar to that of conventional LAMP assay with respect to its sensitivity (up to 100 fg), rapidity (30 min), and specificity. This platform could serve as a prototype for developing on-field diagnostic kits to be used at the point of care centers for the rapid diagnosis of M. oryzae and S. oryzae in rice seeds. This is the first study to report a LAMP-based foldable microdevice platform to detect any plant pathogens.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60 °C for 30 min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100 fg of template DNA compared to 1 ng and 10 pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

First report of Alternaria alternata causing leaf blight on little millet (Panicum sumatrense) in India.

Little millet (LM) is a minor cereal crop grown in the Indian sub-continent. During October 2018, dark brown, circular to oval necrotic spots surrounded by concentric rings were observed on the upper leaf surface of the LM (cv. VS-13) grown in the fields of the University of Agricultural Sciences, Bengaluru, India (13.0784oN, 77.5793oE). As the disease progressed, infected leaves became blighted. Disease incidence up to 53% was recorded in 3 fields of 0.4-hectare area each. Thirty symptomatic leaves were collected to isolate the associated causal organism. The margins of diseased tissue were cut into 5 × 5-mm pieces, surface-sterilized in 75% ethanol for 45 seconds followed by 1% sodium hypochlorite for 1 min, finally rinsed in sterile distilled water five times and placed on PDA. After 7 days of incubation at 25°C, greyish fungal colonies appeared on PDA. Single-spore isolations were performed to obtain ten isolates. Pure cultures of the fungus initially produced light gray aerial mycelia that later turned to dark grey. All isolates formed obclavate to pyriform conidia measured 22.66-48.97µm long and 6.55-13.79µm wide with 1-3 longitudinal and 2-7 transverse septa with a short beak (2.55-13.26µm) (n=50). Based on the conidial morphology, the fungus was identified as Alternaria sp. Further, the taxonomic identity of all ten isolates was confirmed as A. alternata using species-specific primers (AAF2/AAR3, Konstantinova et al. 2002) in a PCR assay. Later, one of the isolate UASB1 was selected, and its internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (gapdh), major allergen Alt a 1 (Alt a 1), major endo-polygalacturonase (endoPG), OPA10-2, and KOG1058 genes were amplified in PCR (White et al. 1990; Berbee et al. 1999; Woudenberg et al. 2015), and the resultant products were sequenced and deposited in the NCBI GenBank (ITS, MN919390; gapdh, MT637185; Alt a 1, MT882339; endoPG, MT882340; OPA10-2, MT882341; KOG1058, MT882342). Blastn analysis of ITS, gapdh, Alt a 1, endoPG, OPA10-2, KOG1058 gene sequences showed 99.62% (with AF347031), 97.36% (with AY278808), 99.58% (with AY563301), 99.10% (with JQ811978), 99.05% (with KP124632) and 99.23% (with KP125233) respectively, identity with reference strain CBS916.96 of A. alternata, confirming UASB1 isolate to be A. alternata. For pathogenicity assay, conidial suspension of UASB1 isolate was spray inoculated to ten healthy LM (cv. VS-13) plants (45 days old) maintained under protected conditions. The spore suspension was sprayed until runoff on healthy leaves, and ten healthy plants sprayed with sterile water served as controls. Later, all inoculated and control plants were covered with transparent polyethylene bags and were maintained in a greenhouse at 28±2 ◦C and 90% RH. The pathogenicity test was repeated three times. After 8 days post-inoculation, inoculated plants showed leaf blight symptoms as observed in the field, whereas no disease symptoms were observed on non-inoculated plants. Re-isolations were performed from inoculated plants, and the re-isolated pathogen was confirmed as A. alternata based on morphological and PCR assay (Konstantinova et al. 2002). No pathogens were isolated from control plants. There is an increasing acreage of LM crop in India, and this first report indicates the need for further studies on leaf blight management and the disease impacts on crop yields.

Comparative genomics of rice false smut fungi Ustilaginoidea virens Uv-Gvt strain from India reveals genetic diversity and phylogenetic divergence.

False smut disease of rice caused by Ustilaginoidea virens, is an emerging threat to rice cultivation worldwide due to its detrimental effects on grain yield and quality. False smut disease severity was 4.44‒17.22% during a roving survey in Kharif 2016 in the four different rice ecosystems of Karnataka, India. Further, 15 pathogen isolates representing four different ecosystems were studied for their virulence and morphometric diversity. Among the 15 strains studied, most virulent strains Uv-Gvt was selected for whole genome sequencing in Illumina NextSeq 500 platform using 2 × 150 bp sequencing chemistry. The total assembled genome of Uv-Gvt was 26.96 Mb, which comprised of 9157 scaffolds with an N50 value of 15,934 bp and 6628 protein-coding genes. Next, the comparative genomic study revealed a similar gene inventory as UV-8b and MAFF 236576 strains reported from China and Japan, respectively. But, 1756 genes were unique to Uv-Gvt strain. The Uv-Gvt genome harbors 422 putative host-pathogen interacting genes compared to 359 and 520 genes in UV-8b and MAFF 236576 strains, respectively. The variant analysis revealed low genetic diversity (0.073‒0.088%) among U. virens strains. Further, phylogenetic analysis using 250 single copy orthologs genes of U. virens revealed a distinct phylogeny and an approximate divergence time. Our study, report the genomic resource of rice false smut pathogen from India, where the disease originated, and this information will have broader applicability in understanding the pathogen population diversity.