Ajay Kumar Mishra

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.

Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiae associated with taro leaf blight.

The Oomycetous fungus Phytophthora colocasiae causing leaf blight of taro is widely distributed in India. Wide geographic range or sexual recombination provides genetic differentiation within this species. To determine how genetic variation is partitioned in P. colocasiae, 14 isolates were isolated from different regions of India, where the incidence of leaf blight is great. Molecular and biochemical techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae. Seven polymorphic enzyme systems revealed 23 isozyme patterns, each uniquely characterised by the presence or absence of electromorphs. Further, 10 oligodeoxynucleotide primers were selected for random amplified polymorphic DNA (RAPD) assays, which resulted in 123 polymorphic bands for 10 isolates of P. colocasiae. The data were entered into a binary matrix and a similarity matrix was constructed using a DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using a NTSYS computer program. Shannons index was used to partition genetic diversity. Similarly, isozymes and RAPDs yielded high estimates of genetic variability. Genetic diversity estimates via isozyme and RAPD pattern indicated 78.26% and 100%, respectively, total diversity among populations. This type of genetic variation in P. colocasiae indicates that variation due to asexual and/or possibly infrequent sexual mechanisms is possible and that genetic differentiation has taken place as a result of geographic isolation. The presence of larger than expected RAPD variation in isolates of P. colocasiae and the presence of distinct different zymotypes among these isolates suggests that genetic recombination (or less likely hybridisation) is at least possible in this fungus and that geographic differentiation has taken place. Even isolates obtained from the same habitat have different RAPD patterns, indicating that many populations of this fungus are made up of more than one genet and that few are derived clonally.

Purification and characterization of elicitor protein from Phytophthora colocasiae and basic resistance in Colocasia esculenta

An elicitor was identified in the fungus Phytophthora colocasiae. The molecular weight of the purified elicitor was estimated by means of gel filtration chromatography and SDS-PAGE and was estimated as 15kDa. Protease treatment severely reduced its activity, allowing the conclusion that the elicitor is proteinaceous. Infiltration of a few nanograms of this proteinaceous elicitor into taro leaves caused the formation of lesions that closely resemble hypersensitive response lesions. The elicitation of the cells was effective in the induction of the activity of lipoxygenase. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, after the infiltration of the elicitor protein. After few days, systemic acquired resistance was also induced. Thus, taro plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence. The obtained results give important information regarding the plant-pathogen interactions, mainly as subsidy for taro improvement against Phytophthora 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.

Cloning and characterization of cDNA encoding an elicitor of Phytophthora colocasiae

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. A cDNA encoding elicitor, the major secreted extracellular glycoprotein of Phytophthora colocasiae, a pathogen of taro (Colocasia esculenta) plants, was isolated, sequenced and characterized. The expression of the corresponding elicitor gene during the disease cycle of P. colocasiae was analyzed. Elicitor was shown to be expressed in mycelium grown in culture media, whereas it was not expressed in sporangiospores and zoospores. In planta, during infection of taro, particularly during the biotrophic stage, expression of elicitor was down-regulated compared to in vitro. The highest levels of expression of elicitor were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The elicitation of the suspension-cultured taro cells was effective in the induction of the enzyme activity of l-phenylalanine-ammonia lyase, peroxidase and lipoxygenase as well as the expression of defense-related endochitinase gene. All these biological activities were exerted within a low concentration range. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in taro plant against Phytophthora leaf blight.