Muthulekshmi Lajapathy Jeeva

A species-specific polymerase chain reaction assay for rapid and sensitive detection of Sclerotium rolfsii

The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. The timely and accurate detection of pathogens is critical in the study of epidemiology and management of plant diseases. A polymerase chain reaction (PCR)-based method was developed for the identification and detection of Sclerotium rolfsii in Amorphophallus paeoniifolius plants. A PCR primer pair specific for S. rolfsii was designed based on the sequence of the internal transcribed spacer region. The designed primer pair SCR-F/SCR-R amplified a 540-bp product from S. rolfsii DNA and did not amplify DNA from A. paeoniifolius or several other fungi pathogenic to A. paeoniifolius. In conventional PCR, the limit of detection of pure fungal gDNA was 6 pg ml−1, which was reduced 2-fold within a plant DNA background. S. rolfsii DNA was detected in inoculated A. paeoniifolius and 12 h after inoculation in symptomless tuber samples. The protocol was assessed for the detection of S. rolfsii in infected soils.

Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real‐time PCR assay

Conventional and real-time PCR assays were developed for sensitive and specific detection of Phytophthora colocasiae, an oomycete pathogen that causes leaf blight and corm rot of taro. A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. In conventional PCR, the lower limit of detection was 50 pg DNA, whereas in real-time PCR, the detection limit was 12.5 fg for the primer based on Ypt1 gene. The cycle threshold values were linearly correlated with the concentration of the target DNA (range of R(2) = 0.911-0.999). All the primer sets were successful in detecting P. colocasie from naturally infected leaves and tubers of taro. Phytophthora colocasiae was detected from artificially infested samples after 18 and 15 h of postinoculation in conventional and real-time PCR assay, respectively. The developed PCR assay proved to be a robust and reliable technique to detect P. colocasiae in taro planting material and for assessing the distribution of pathogen within fields, thus aid in mitigating taro leaf blight.

Characterisation of Phytophthora colocasiae isolates associated with leaf blight of taro in India

Leaf blight and corm rot caused by Phytophthora colocasiae are the most devastating diseases of taro. Fourteen P. colocasiae isolates collected from different states of India were characterised for mating type, metalaxyl fungicide sensitivity and aggressiveness on taro. These organisms were identified as P. colocasiae based on morphological characteristics, internal transcribed spacer (ITS) sequence homology and pathogenicity to taro plants. Ribosomal DNA (rDNA) ITS regions of P. colocasiae isolates were examined, and compared with additional Phytophthora species. All isolates of P. colocasiae fell within a single cluster in phylogenetic trees, regardless of their geographic origins. These isolates were more closely related to Phytophthora capsici. All isolates except the isolate collected from Sikkim state of India (98–111) were sensitive to metalaxyl at 100 ppm and it was also scored for causing higher average foliar disease and tuber rot ratings. Isolates tested exhibited four growth patterns in cultures: cottony, rosaceous, petaloid and stellate. P.colocasiae isolates, including an IISR isolate (PC-73), with cottony growth pattern did not grow at 36°C. The mean oospore diameter of A1 mating type isolates was greater than that of A2 mating type isolates. Six of 14 isolates tested produced chlamydospores in V8-CaCO3 liquid medium.

Rapid and sensitive detection of potyvirus infecting tropical tuber crops using genus specific primers and probes

A reverse transcription polymerase chain reaction assay using potyvirus specific primers designed from the core of the coat protein was carried out, and a cDNA fragment of 327 bp was obtained from most of the potyviruses infecting the tropical tuber crops. Reverse transcription polymerase chain reaction (RT-PCR) products were sequenced and found to be derived from the expected virus. Specific cDNA probe was generated from the amplicon, and the probe was then successfully used for the diagnosis of the potyviruses infecting the major tuber crops through biotinylated nucleic acid spot hybridisation. The specific probe developed could detect the potyviruses infecting tuber crops namely SPFMV, DsMV and DAV from sweet potato, aroids and yams respectively.

Rock inhabiting potassium solubilizing bacteria from Kerala, India: characterization and possibility in chemical K fertilizer substitution.

The role of rock inhabiting bacteria in potassium (K) solubilization from feldspar and their application in crop nutrition through substitution of fertilizer K was explored through the isolation of 36 different bacteria from rocks of a major hill station at Ponmudi in Thiruvananthapuram, Kerala, India. A comprehensive characterization of K solubilization from feldspar was achieved with these isolates which indicated that the K solubilizing efficiency increases with decrease in pH and increase in viscosity and viable cell count. Based on the level of K solubilization, two potent isolates were selected and identified as Bacillus subtilis ANctcri3 and Bacillus megaterium ANctcri7. Exopolysaccharide production, scanning electron microscopic and fourier transform infrared spectroscopic studies with these efficient strains conclusively depicted the role of low pH, increase in viscosity, and bacterial attachment in K solubilization. They were also found to be efficient in phosphorus (P) solubilization, indole acetic acid production as well as tolerant to wide range of physiological conditions. Moreover, the applicability of K containing rock powder as a carrier for K solubilizing bacteria was demonstrated. A field level evaluation on the yield of a high K demanding tuberous vegetable crop, elephant foot yam (Amorphophallus paeoniifolius (dennst.) nicolson) established the possibility of substituting chemical K fertilizer with these biofertilizer candidates successfully.

Molecular detection and identification of Dasheen mosaic virus infecting Amorphophallus paeoniifolius

Occurrence of various types of viral symptoms viz. mosaic, mottling, puckering, stunting and filiformy/shoestring was noticed on Elephant foot yam in India. Immunosorbent electron microscopy of infected leaf samples with Potyvirus group–specific antiserum revealed the association of flexuous Potyvirus particles. Infected leaf samples were collected from different locations in Kerala, Orissa and Andhra Pradesh; and total RNA was extracted from infected leaves. One-step reverse transcription polymerase chain reaction assay was carried out using Potyvirus group–specific primers and an amplified product of 327 and 719 bp were obtained. Sequence analysis of nucleic acid and deduced amino acid sequences with the already available Potyvirus sequences in Genbank showed 91% and 97% similarity with Dasheen mosaic virus (DsMV) isolate DeSLK2 (accession Number AJ305434) at nucleic acid and amino acid level respectively. Comparison of 3′UTR region using universal primers MJ1 and M4T also confirmed that the virus under study is DsMV and is closely related to DeSLK2 isolate. This is the first molecular level detection of the virus infecting Amorphophallus paeoniifolius in India.

Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro

Leaf blight caused by Phytophthora colocasiae is the most destructive disease affecting taro (Colocasia esculenta) worldwide including India. Fungicides (primarily metalaxyl) remain as an important strategy to manage taro leaf blight in India over decades. It is important to monitor isolate sensitivity to identify build-up of fungicide resistance and thereby modify fungicide usage strategies. P. colocasiae isolates representing four different geographical regions of India were evaluated for their sensitivity to metalaxyl and three other commercially available fungicides viz. Samarth, Biofight and Akoton by poisoned media technique. All the isolates tested were sensitive to metalaxyl, nevertheless there is an increase in the effective concentration compared to the previous reports. Among the other fungicides, Samarth was found to be superior in completely inhibiting mycelial growth at 0.05% followed by Biofight at 1%. Metalaxyl and Akoton® shared a common inhibitory concentration at 2%. The most effective fungicide determined by the in vitro method was evaluated in vivo for studying the pattern of inhibition before and after the disease development in detached taro leaf. The results of the study revealed that build-up on metalaxyl resistance in P. colocasiae is in its course and caution should be taken while administering against taro leaf blight. Fungicide Samarth could be used as an alternative to metalaxyl for management of taro leaf blight.

Inhibitory activity of plant growth regulators on Phytophthora palmivora causing cassava tuber rot

Auxins and cytokinins are implicated in a wide variety of developmental and physiological processes in plants. Phytophthora palmivora causes tuber rot in cassava growing regions of Tamil Nadu and Kerala, South India. The in vitro effect of cytokinin, benzyl amino purine (BAP) and auxins, naphthalene acetic acid (NAA) and indole acetic acid (IAA) on P. palmivora mycelium growth was investigated. The inhibitory activity varied among the growth regulators and complete inhibition of the pathogen was observed at 50, 2000 and 2500 ppm by the BAP, IAA and NAA, respectively. The effective growth regulator, BAP was also analysed on tubers before and after the invasion of the pathogen to observe its effect in tuber. Further, it was also checked against the bio-control agent Trichoderma harzianum. The study indicates that the use of BAP could be an important approach in controlling tuber rot pathogen, P. palmivora.

Morphological, pathological and molecular characterization of Phytophthora colocasiae responsible for taro leaf blight disease in India

The oomycetous fungus Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro and is widely distributed in India. A combination of morphological (colony morphology, mating type, pathogenicity, metalaxyl sensitivity) and molecular techniques (rDNA ITS sequencing and Start codon targeted polymorphism, ScoT analysis) was used to characterize 50 isolates of P. colocasiae obtained from different locations in India. Considerable differences in morphological parameters were observed. ScoT analysis revealed high polymorphism among the isolates. This study confirms that isolates of P. colocasiae are highly dynamic in nature and a considerable degree of diversity exists among them. A detailed knowledge of the morphological and molecular characters of P. colocasiae will help in developing suitable control strategies against the taro leaf blight disease.

Incidence and identification of Cassava tuber rot caused by Phytophthora palmivora

The occurrence of Cassava tuber rot in regions of Kolli hills, Kollam, and Kottayam of South India, causes major economic loss up to 70% in Cassava production. The disease tuber is characterised by brown watery lesions with foul smell, making it unfit for further use. The sporangia of the pathogen were oval and ellipsoid with a short pedicle. Identification of the isolate from these regions was also confirmed by ribosomal internal transcribed spacer (ITS) of rDNA region. The pathogen was highly aggressive when pathogenicity was tested. Based on morphological, pathogenicity and ITS sequences, the pathogen was identified as Phytophthora palmivora. Development of integrated disease management practices is essential to combat the disease. This is the first report recording the spread of Cassava tuber rot disease in regions of Kolli hills of Tamil Nadu and Kollam and Kottayam, of Kerala.