Prevalence of groundnut dry root rot (Macrophomina phaseolina (Tassi) Goid.) and its pathogenic variability in Southern India.

Macrophomina phaseolina is the most devastating and emerging threat to groundnut production in India. An increase in average temperature and inconsistent rainfalls resulting from changing climatic conditions are strongly believed to aggravate the disease and cause severe yield losses. The present study aims to conduct a holistic survey to assess the prevalence and incidence of dry root rot of groundnut in major groundnut growing regions of Southern India, viz., Andhra Pradesh, Telangana, Karnataka, and Tamil Nadu. Furthermore, the pathogenic variability was determined using different assays such as morphological, cultural, pathogenic, and molecular assays. Results indicate that disease incidence in surveyed locations ranged from 8.06 to 20.61%. Both temperature and rainfall played a major role in increasing the disease incidence. The pathogenic variability of M. phaseolina isolates differed significantly, based on the percent disease incidence induced on cultivars of JL-24 groundnut and K-6 groundnut. Morphological variations in terms of growth pattern, culture color, sclerotia number, and sclerotia size were observed. The molecular characterization of M. phaseolina isolates done by ITS rDNA region using ITS1 and ITS4 primers yielded approximately 600 bp PCR amplicons, sequenced and deposited in GenBank (NCBI). Molecular variability analysis using SSR primers indicated the genetic variation among the isolates collected from different states. The present investigation revealed significant variations in pathogenic variability among isolates of M. phaseolina and these may be considered important in disease management and the development of resistant cultivars against groundnut dry root rot disease.

First report of association of 'Candidatus Phytoplasma asteris' with Moringa oleifera leaf yellowing and stunting disease in India.

Moringa oleifera (family Moringaceae) also known as the 'drumstick tree' is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain 'Candidatus Phytoplasma asteris' (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with 'Ca. Phytoplasma asteris' (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B ('Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Complete genome sequence and phylogenetic relationships of tobacco streak virus causing groundnut stem necrosis disease in India.

Tobacco streak virus (TSV, genus Ilarvirus family Bromoviridae) is known to cause stem necrosis disease (SND) in groundnut (Arachis hypogaea) since 2000 in Southern India. The TSV isolate infecting groundnut so far has not been characterized based on the complete genome sequence. In this study, TSV was isolated from a naturally infecting groundnut plant in Kadiri, the hot-spot of the SND in southern India. During the Kharif season of 2014, groundnut plants in an experimental field were affected with chlorosis and necrosis in leaf, stem and buds. The cent percent of the 48 samples with these symptoms collected from the field tested positive for TSV in ELISA samples in this context. One isolate, GN-Kad was established from a single lesion on cowpea cv. C-152 through successive sap inoculation. Cloning and sequencing of coat protein gene (717 nucleotides) of the isolate showed high sequence identity (98-99%) with the TSV isolates reported from different crops in India. The isolate produced local necrotic rings or veinal necrosis following sap inoculation to cowpea (cultivars C-152, Pusa Komal, Pusa Sukomal and Krishi Kanchan), French bean and sunflower; whereas, it produced systemic chlorotic mottling symptoms in Nicotiana benthamiana. The three segments of the virus genome (RNA 1, RNA 2 and RNA 3) contained 3523, 2903 and 2232 nucleotides, respectively. The overall genome sequence (8639 nt) of the present isolate shared 77-99% of nucleotide sequence identity with that of the other seven isolates reported from Australia, India and USA. The GN-Kad shared very close phylogenetic relationship with the okra and pumpkin isolates reported from India. The present report is the first comprehensive study of the molecular characterization of TSV associated with the stem necrosis disease of groundnut.

First Report of Tobacco streak virus Infecting Pigeon Pea (Cajanus cajan) in India.

Pigeon pea is an important pulse crop grown in diversified cropping systems in India. In the rainy season of August 2011 and September 2012, pigeon pea cv. LRG 30 plants with leaf necrosis having wrinkled margin on one side were observed in Kadiri mandal of the Anantapuram district of Andhra Pradesh (A.P.), India. Symptoms included necrotic spots on young leaves followed by wilting of leaves, petiole and branch/axillary shoot proliferation, with small leaves having mosaic symptoms. Symptomatic leaves were sap-inoculated onto 10 seedlings of cowpea (cv. Pusa Komal) using 0.01 M phosphate buffer (pH 7.0). Localized necrotic lesions developed in all the inoculated plants after 2 days post inoculation. Field symptoms were reproduced on healthy pigeon pea upon back inoculation using single lesions of infected cowpea leaves. In direct antigen coating (DAC)-ELISA, all the infected pigeon pea and cowpea leaf samples were positive to a polyclonal antiserum specific to Tobacco streak virus (TSV) supplied by ICRISAT, India. Total RNA was extracted using infected pigeon pea and healthy leaf samples by TRI Reagent (Sigma). Reverse transcription (RT)-PCR was carried out using primers specific to the coat protein (CP) gene of TSV (1). A product of the 700-bp DNA fragment was obtained in field-infected pigeon pea samples but not in healthy controls. The amplicon was cloned into PTZ57R/T using the Ins TA clone PCR kit (Fermentas). Recombinant clone was sequenced in both directions and the CP gene sequence obtained was deposited in GenBank (KF220492). Sequence analysis of the CP gene of TSV from pigeon pea shared 98 to 100% identity with Indian TSV isolates originating from different hosts including groundnut (FJ355948), mung bean (FJ749259), and sunflower (DQ864448), and 88 to 92% similarity with TSV type isolate (white clover: X00435) both at nucleotide and amino acid levels. TSV belongs to the genus Ilarvirus of family Bromoviride and has a wide host range. TSV is pollen borne, assisted by thrips causing mechanical injury (2). To our knowledge, this is the first report of TSV on pigeon pea in India and was widespread in Anantapuram, Kadapa, Kurnool, and Mahbubnagar districts of A.P. Yield loss depends on the stage of infection as early infection resulted in complete failure of the crop. TSV was prevalent on many legume crops such as black gram, green gram, and groundnut in A.P, Tamil Nadu, Karnataka, and Maharashtra states (3). TSV infection of pigeon pea may pose a serious implication for pulse production. References: (1). A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2). M. Sharman et al. Australian Plant Dis. 6:54, 2011. (3). K. Vemana and R. K. Jain. Indian J. Virol. 21:117, 2010.

Comparative reaction of popular high yielding spanish and virginia bunch groundnut varieties to Tobacco streak virus.

Reaction of five spanish (JL 24, TMV 2, Kadiri 6, Kadiri 9 and Anantha) and a virginia (Kadiri 7 Bold) groundnut cultivars was studied against Tobacco streak virus (TSV) by sap inoculation using different age group of plants [7-84 days after sowing (DAS)]. Among different cultivars, incubation period varied from 4 to 28 days and high incubation period recorded in Kadiri 7 Bold. The percent infection decreased with increase in the age of the plants in all the cultivars as it ranged up to 100 % in both pre-flowering (7-21 DAS) and flowering stage (28-63 DAS) plants followed by 50-100 % in maturity stage plants (70-84 DAS) except Kadiri 7 Bold. Similarly, 100 % wilting was observed in pre flowering stage plants against no wilting in maturity stage plants. However, cultivars differed in per cent wilting of flowering stage plants by recording maximum wilting (100 %) in JL24, Kadiri 6 and minimum (25.0 %) in Kadiri 7 Bold. Both localized (necrotic spots, veinal necrosis) and systemic (petiole necrosis, necrotic spots on young leaves, top growing bud and stem necrosis, axillary shoot proliferation, stunting, peg necrosis, pod necrosis, wilting of plant) symptoms induced by TSV were similar among all cultivars without any new symptoms. Prolonged stage of axillary shoot proliferation was observed for the first time in all the cultivars. In maturity stage plants of Kadiri 7 Bold, Kadiri 9 and Anantha, systemic symptoms restricted to leaf and petiole necrosis only. Virus titer varied significantly with the age of plants and inoculum harvest at days post inoculation and least virus titer recorded by Kadiri 7 Bold at all stages of infection. Among different cultivars, Kadiri 7 Bold was least susceptible/tolerant to TSV by registering higher incubation period with less per cent infection, wilt and titer.

New Experimental Hosts of Tobacco streak virus and Absence of True Seed Transmission in Leguminous Hosts.

Of 70 plant species tested, 50 species were susceptible to Tobacco streak virus (TSV) on sap inoculation. Both localized (necrotic and chlorotic spots) and systemic (necrotic spots, axillary shoot proliferation, stunting, total necrosis and wilt) symptoms are observed by majority of plant species. Eleven new experimental hosts were identified viz., Amaranthus blitum var. oleracea (Chaulai sag), Celosia cristata (Cocks comb), Beta vulgaris var. bengalensis (Palak/Indian spinach), Calendula officinalis (Pot marigold), Chrysanthemum indicum, Cosmos sulphurens (Yellow cosmos), Citrullus lunatus (Watermelon), Lagenaria siceraria (Bottle gourd), Coriandrum sativum (Coriander), Hibiscus subderiffa var. subderiffa (Roselle) and Portulaca oleraceae (Little hogweed). Detected groundnut seed infection with TSV for the first time by Direct antigen coated immunosorbent assay (DAC-ELISA) using whole seed. The seed infection ranged from 18.9 to 28.9% among the seeds collected from naturally infected and sap inoculated groundnut varieties (JL 24, TMV 2, Prasuna, Kadiri 6, Kadiri 9, Anantha and Kadiri 7 Bold) belonging to spanish and virginia types. Further, TSV was detected both in pod shell and seed testa and none of the samples showed the presence of TSV either in cotyledon or embryo. Grow-out and bio-assay tests proved the absence of seed transmission in groundnut and other legume crops. Hence, TSV isolate was not a true seed transmission case under Indian conditions in legumes.