Morphological and molecular intervention in identifying Phytophthora spp. causing leaf and nut fall in nutmeg (Myristica fragrans Houtt.)

Abstract

Nutmeg, a major crop of India provides two important spices. During the monsoon season of 2011, a serious leaf and nut fall occurred in nutmeg plantations and Phytophthora ramorum was reported as the causal organism. P. ramorum being an alien species, the aetiology warranted a detailed investigation. Phytophthora isolated from different endemic locations were studied for morphological and molecular characterization using ITS, ITS-RFLP, MLST, SSCP and hybrid analysis. The isolates showed papillate, ovoid-obovoid sporangia with pedicel length > 20 μm which is distinct from the semi-papillate, caducous sporangia with short pedicel of <5 μm characteristic of P ramorum. The isolates failed to grow at 10 °C or at 37 °C and chlamydopsores were not produced in culture, a characteristic distinct to P. ramorun. MLST analysis placed the isolates in Clade 2 while P. ramorum belongs to Clade 8. P. ramorum has a unique set of morphological characteristics and a unique molecular sequence at the ITS region which delineate it from other Phytopthora species. Among the eight isolates studied, ITS region of six isolates (13–01–13-06) showed similarity to P. meadii and two isolates (13–55 and 98–68) to P. tropicalis. In SSCP, the isolates formed a separate group distinct from P.meadii of cocoa and cardamom. The double peaks in the ITS chromatograms indicated hybridity and further hybrid analysis placed them under P. citrophthora and P. meadii (Cit-Med) lineage. Thus due to the distinct differences from P. ramorum and evidences of shared lineage with P. citrophthora and P. meadii, we propose to call these isolates as P. citromeadii (13–01- 13-06) and P. citrocaptalis (13–55 and 98–68).