METHODS: Thirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.
RESULTS: Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5' and 3' regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5' and 3' regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.
CONCLUSIONS: High genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5' and the 3' regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.
METHODS AND RESULTS: Based on the sequences of internal transcribed spacer (ITS), TEF1-α and β-tubulin, as well as on the phylogenetic analysis of combined sequences, four species of Lasiodiplodia (L. theobromae,L. pseudotheobromae, L. iranensis, L. mahajangana) and two species of Neofusicoccum (N. ribis, N. parvum) were identified. Pseudofusicoccum violaceum, Neoscytalidium dimidiatum and three species of Botryosphaeria (B. scharifii, B. dothidea, B. ramosa) were identified based on sequences of ITS and TEF1-α. Pathogenicity test of selected isolates were tested on Chok Anan, Waterlily and Falan mango cultivars. Generally, all species were observed to be pathogenic on the three tested mango cultivars on wounded fruits, except for N. ribis and N. parvum, which were pathogenic on both wounded and unwounded fruits. However, N. ribis was only pathogenic on cultivar Falan, whereas B. ramosa were pathogenic on cultivars Waterlily and Falan.
CONCLUSIONS: Eleven species of Botryosphaeriaceae were associated with mango stem-end rot in Malaysia. To the best of our knowledge, four species, namely L. mahajangana, B. ramosa, N. ribis and P. violaceum are the first recorded Botryosphaeriaceae fungi associated with stem end rot of mango.
SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of Botryosphaeriaceae fungi is important to establish suitable control measures and quarantine requirements. Many species have a wide host range, which means that there is a possibility of cross infection from other infected plants.