Root lesion nematodes on potato in Western Australia, and assessing candidate genes of Pratylenchus penetrans for control using RNAi

Saiful Islam

Potato is Australia's most economically important horticultural crop, worth $830.2 million per annum, and the second most important horticultural crop in Western Australia (WA) valued at $177 million pa. Potato yields can be reduced by up to 50% from infestation by root-lesion nematodes (RLNs, Pratylenchus spp.); infestation also affects tuber quality and marketability. Identifying which RLN species is present in potato fields is the first step in finding appropriate control strategies. One such new control strategy is RNA interference (RNAi), a naturally occurring gene regulation mechanism in eukaryotes, which can be used to identify and silence vital genes, including those required for parasitism, such as by plant parasitic nematodes (PPNs) like Pratylenchus spp. The first aspect of the research undertaken was to investigate how widespread Pratylenchus spp. are in the potato-growing regions of WA. Morphological and molecular analyses of nematodes collected from eleven paddocks in four potato production regions indicated that P. penetrans was widespread and the most abundant Pratylenchus species present. Attraction and penetration assays showed that they were more attracted to and penetrated the roots of potato cv Desiree than cv Atlantic. P. penetrans was also more common in the roots of both cultivars than P. neglectus (p < 0.05). The reproduction factors in cv. Desiree for each nematode was more than one, indicating that both could thrive and reproduce well in this potato cultivar. Five target genes expressed in oesophageal gland cells of P. penetrans, some of which might encode effector proteins were chosen for further study. The expression of the five target genes (Pp-256, Pp-066, Pp-504, Pp-972 and Pp-480) in eggs, juveniles and adult stages of P. penetrans was assessed, and the expression of target genes was highest in adults whereas it was lowest in eggs. The expression of Pp-066, a gene of unknown function, was the highest in adults, while the expression of Pp-504 was the lowest. The target genes were then studied by soaking in dsRNA to determine whether they could be useful candidates for RNAi control of P. penetrans. Soaking of mixed stages of P. penetrans in dsRNA for 16 hours to induce transient RNAi of the five genes changed nematode behaviour, movement, infectivity and reproduction, indicating that these expression of these genes was required for nematode growth or development. Down-regulation of the transcripts by RNAi was confirmed using semi-quantitative PCR, with gene Pp-504, being the most markedly down-regulated. Attraction of dsRNA-treated nematodes to host roots was reduced by up to 25%, the most significant reduction was for nematodes after RNAi of Pp-504, with gene Pp-066 (18%) the lowest. Potato plants in soil were inoculated with dsRNA-treated nematodes, and after 42 days, reproduction was reduced between 55.3 and 70.2%. The nematodes treated with the dsRNA of the gene Pp-504 were the most affected. Constructs to transform potato plants to express dsRNA to the target genes constitutively using Host-Induced Gene Silencing (HIGS) were made and are ready for use in a subsequent project. Overall, the results suggest that all five genes are important for nematode infectivity, survival or reproduction, and hence could be candidates for RNAi control of P. penetrans. The results also add to the potential to develop RNAi technology to control P. penetrans, either using Host Induced Gene Silenicng (HIGS) or the developing strategy of Spray-Induced Gene Silencing (SIGS).

Root lesion nematodes on potato in Western Australia, and assessing candidate genes of Pratylenchus penetrans for control using RNAi

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