Isolation and identification of fungi associated with wilt, rot and anthracnose diseases in Vanilla planifolia in Pahang and Sabah, Malaysia

Fungal diseases had caused significant loss in vanilla productions as several species have been found associated with wilt, rot and anthracnose diseases. In this study, infected vanilla plant parts were collected from three vanilla farms in Pahang and Sabah, Malaysia. Tissue transplanting techniques was practised to grow the fungi. The cultural and morphological characteristics of pure culture such as the colony size, colour and conidia characters were observed. The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA were replicated in Polymerase Chain Reaction (PCR) using fungal universal primers, ITS1 and ITS4 to verify this morphological identification. Pathogenicity of these isolated fungus was tested on fresh mature vanilla leaf tissue, and antagonistic test was conducted to screen the ability of four Trichoderma species; T. harzianum, T. viride, T. koningii and T. virens as biological control agents (BCAs) to suppress the growth of the isolates in plate assay experiment. The Percentage Inhibition Radial Growth (PIRG) analysis was used to evaluate the efficacy of BCAs against isolated fungus. A total of 22 fungal isolates were recovered during the isolation from the disease vanilla parts in 2015 and 2016. Two different fungal groups, Fusarium species and Colletotrichum species were identified based on cultural and morphological observation. The sequencing DNA of these isolates was compared against the ex-type strains sequences from the NCBI Genbank data. Fusarium species comprises the largest percentage of isolates recovered ranked from F. fujikuroi (7 isolates) which acted as the most dominant species followed by F. oxysporum (6 isolates), Fusarium sp. (2 isolates), F. proliferatum (2 isolate) and F. solani (1 isolate). There were four isolates identified as Colletotrichum gloeosporioides. Phylogenetic study constructed had grouped these two genus of Fusarium and Colletotrichum into similar clusters but different subclusters. Fusarium complex species such as Fusarium sp., F. proliferatum and F. fujikuroi were grouped into similar subclades. Pathogenicity test reveals significant differences (p<0.001) in the pathogenic capacity of the isolates with F. oxysporum and C. gloeosporiodes provided high pathogenic that caused a high level of damage (3=rot and 4=tissue death) with p<0.01. Other Fusarium species were considered as less pathogenic to non-pathogenic to vanilla. PIRG analysis shows high antagonistic activity (PIRG>50) was demonstrated by T. koningii on F. oxysporum, F. proliferatum, F. solani and F. fujikuroi. This study includes valuable information on the fungi associated with wilt, rot and anthracnose disease in vanilla grown in Malaysia. The data collection of Fusarium species associated with vanilla; in particular for both pathogenic and non-pathogenic Fusarium species will contribute to future development on disease management.