Sympatric occurrence of sibling Phytophthora species associated with foot rot disease of black pepper in India.

Foot rot disease caused by Phytophthora capsici is a serious threat to black pepper cultivation in India and globally. High diversity exists among the Phytophthora isolates of black pepper and hence detailed investigations of their morphology and phylogenetic taxonomy were carried out in the present study. In order to resolve the diversity, 182 isolates of Phytophthora, collected from different black pepper-growing tracts of South India during 1998-2013 and maintained in the National Repository of Phytophthora at ICAR-Indian Institute of Spices Research, Kozhikode, were subjected to morphological, molecular and phylogenetic characterization. Morphologically all the isolates were long pedicellate with umbellate/simple sympodial sporangiophores and papillate sporangia with l/b ranging from 1.63 to 2.55 µm. Maximum temperature for the growth was ~ 34 °C. Chlamydospores were observed in "tropicalis" group, whereas they were absent in "capsici" group. Initial molecular studies using internal transcribed spacer (ITS) marker gene showed two clear cut lineages-"capsici-like" and "tropicalis-like" groups among them. Representative isolates from each group were subjected to host differential test, multilocus sequence typing (MLST) and phylogeny studies. MLST analysis of seven nuclear genes (60S ribosomal protein L10, beta-tubulin, elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA and TigA gene fusion protein) clearly delineated black pepper Phytophthora isolates into two distinct species-P. capsici and P. tropicalis. On comparing with type strains from ATCC, it was found that the type strains of P. capsici and P. tropicalis differed from black pepper isolates in their infectivity on black pepper. The high degree of genetic polymorphism observed in black pepper Phytophthora isolates is an indication of the selection pressure they are subjected to in the complex habitat which ultimately may lead to speciation. So based on the extensive analysis, it is unambiguously proved that the foot rot disease of black pepper in India is predominantly caused by two species of Phytophthora, viz. P. capsici and P. tropicalis. Presence of multiple species of Phytophthora in the black pepper agro-ecosystem warrants a revisit to the control strategy being adopted for managing this serious disease. The silent molecular evolution taking place in such an ecological niche needs to be critically studied for the sustainable management of foot rot disease.

Association of Lasiodiplodia theobromae with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses.

Lasiodiplodia theobromae is a cosmopolitan pathogen geographically widespread in tropics and subtropics inciting economically important diseases on diverse plant genera. In the present study, Lasiodiplodia theobromae associated with nutmeg exhibiting die-back and declining symptoms was identified and characterized by adopting a polyphasic approach. The disease was characterized with the symptoms including general decline, water-soaking patches on branches and tree trunk, die-back of branches, necrotic lesions beneath water-soaked lesions and necrosis of vascular tissues. The isolates representing diverse nutmeg growing tracts were initially identified as Lasiodiplodia species based on macro- and micro-morphological characteristics. Subsequent analyses of internal transcribed spacer (ITS), partial elongation factor 1-alpha (EF1-α) and ß-tubulin (ß-tub) genes identified the pathogen as Lasiodiplodia theobromae. Pathogenicity studies were proved on nutmeg twigs and branches (in vitro) as well as on saplings (in vivo). The present investigation enunciated the association of Lasiodiplodia theobromae with die-back and decline of nutmeg employing a polyphasic approach which warrants further investigations on its spatio-temporal distribution, pathogen diversity, weather-host-pathogen interaction and formulating prospective disease management strategies.

Efficacy of bioagents against Pythium deliense Meurs associated with yellowing of black pepper.

Yellowing and wilting of black pepper vines is a serious concern in many black pepper growing tracts where Pythium deliense was recently emerged as a pathogen from the rhizosphere of affected vines, which is proved to be pathogenic by Koch's postulates. As a measure to manage the symptoms, bioagents were evaluated against infection by P. deliense. Among the seven bioagents tested, Trichoderma harzianum and Streptomyces albulus showed 100% inhibition in vitro followed by one Streptomyces sp. and S. rimosus (75.33%). The potential ones were further evaluated under the hydroponic system in vivo by challenge inoculation. No root infection was noticed with T. harzianum and S. albulus inoculation, instead, the inoculated plants showed root regeneration. This suggests the efficiency of these bioagents on plant growth promotion as well as on disease suppression. Biochemical analysis of the hydroponic medium showed an increase in membrane conductivity in all the treatments except in T. harzianum. The release of phenolic compounds into the medium was lowest with T. harzianum indicating the prevention of pathogen invasion. In planta evaluation under greenhouse condition and field evaluation also showed the protective effect of T. harzianum and S. albulus with a reduction in the intensity of yellowing to an extent of 73.1% and 71.2%, respectively. The study revealed that T. harzianum and the actinomycete S. albulus had the potential to prevent the root rot caused by P. deliense.

Phyllosphere-associated Methylobacterium: a potential biostimulant for ginger (Zingiber officinale Rosc.) cultivation.

Methanol, a by-product associated with plant metabolism, is a substrate for pink pigmented facultative methylotrophs (PPFMs) of phyllosphere. The symbiotic interaction of PPFMs has many desirable effects on plant growth and disease resistance. The present study investigated the potential of native PPFMs for mitigating biotic stress and plant growth promotion in ginger. PPFMs were isolated from ginger phyllosphere by leaf imprint technique and screened against major fungal phytopathogens of ginger viz. Macrophomina phaseolina, Sclerotium rolfsii, Pythium myriotylum, Colletotrichum gloeosporioides and Fusarium oxysporum. Among the 60 PPFMs, IISRGPPFM13 was selected for its highly inhibitory activity against the target pathogens. The isolate was useful for mineral solubility, production of IAA, siderophores and hydrolytic enzymes like cellulase, pectinase, lipase, amylase and chitinase. On in planta experiments revealed that IISRGPPFM13 considerably increased plant growth parameters when the bacterium was applied as soil drenching cum foliar spraying. Methanol utilization potential of the isolate was confirmed by mxaF gene analysis where the sequence showing 95.51% identity towards Methylobacterium platani and M. iners. Further, 16S rRNA gene sequence showing 98.73% identity with M. komagatae 002-079 T (AB252201). This is the first report of its kind that a genus of Methylobacterium with biostimulant potential isolated from the phyllosphere of ginger.