Novel Functions of the Fatty Acid and Retinol Binding Protein (FAR) Gene Family Revealed by Fungus-Mediated RNAi in the Parasitic Nematode, Aphelenchoides besseyi

RNA interference (RNAi) is a powerful tool for the analysis of gene function in nematodes. Fatty acid and retinol binding protein (FAR) is a protein that only exists in nematodes and plays an important role in their life activities. The rice white-tip nematode (RWTN), Aphelenchoides besseyi, is a migratory endoparasitic plant nematode that causes serious damage in agricultural production. In this study, the expression levels of eight RWTN genes were effectively decreased when RWTN was fed Ab-far-n (n: 1–8) hairpin RNA transgenic Botrytis cinerea (ARTBn). These functions of the far gene family were identified to be consistent and diverse through phenotypic changes after any gene was silenced. Such consistency indicates that the body lengths of the females were significantly shortened after silencing any of the eight Ab-far genes. The diversities were mainly manifested as follows: (1) Reproduction of nematodes was clearly inhibited after Ab-far-1 to Ab-far-4 were silenced. In addition, silencing Ab-far-2 could inhibit the pathogenicity of nematodes to Arabidopsis; (2) gonad length of female nematodes was significantly shortened after Ab-far-2 and Ab-far-4 were silenced; (3) proportion of male nematodes significantly increased in the adult population after Ab-far-1, Ab-far-3, and Ab-far-5 were silenced, whereas the proportion of adult nematodes significantly decreased in the nematode population after Ab-far-4 were silenced. (4) Fat storage of nematodes significantly decreased after Ab-far-3, Ab-far-4, and Ab-far-7 were silenced. To our knowledge, this is the first study to demonstrate that Ab-far genes affect sex formation and lipid metabolism in nematodes, which provides valuable data for further study and control of RWTNs.

Characterization and Functional Importance of Two Glycoside Hydrolase Family 16 Genes from the Rice White Tip Nematode Aphelenchoides besseyi

The glycoside hydrolase family 16 (GH16) is widely found in prokaryotes and eukaryotes, and hydrolyzes the β-1,3(4)-linkages in polysaccharides. Notably, the rice white tip nematode Aphelenchoides besseyi harbors a higher number of GH16s compared with other plant-parasitic nematodes. In this work, two GH16 genes, namely AbGH16-1 and AbGH16-2, were isolated and characterized from A. besseyi. The deduced amino acid sequences of AbGH16-1 and AbGH16-2 contained an N-terminal signal peptide and a fungal Lam16A glucanase domain. Phylogenetic analysis revealed that AbGH16-1 and AbGH16-2 clustered with ascomycete GH16s, suggesting AbGH16-1 and AbGH16-2 were acquired by horizontal gene transfer from fungi. In situ hybridization showed that both AbGH16-1 and AbGH16-2 were specifically expressed in the nematode gonads, correlating with qPCR analysis that showed the high transcript levels of the two genes in the female nematodes. AbGH16-1 and AbGH16-2 were also significantly induced in nematodes feeding on Botrytis cinerea. Characterization of the recombinant protein showed AbGH16-1 and AbGH16-2 displayed pronounced inhibition of both conidial germination and germ tube elongation of B. cinerea. In addition, silencing of AbGH16-1 and AbGH16-2 by RNA interference significantly decreased the reproduction ability of A. besseyi and had a profound impact on the development process of offspring in this nematode. These findings have firstly proved that GH16s may play important roles in A.besseyi feeding and reproduction on fungi, which thus provides novel insights into the function of GH16s in plant-parasitic nematodes.

Studies on the efficiency of different inoculation methods of rice white-tip nematode, Aphelenchoides besseyi

Summary To fulfil different research purposes, five methods to inoculate Aphelenchoides besseyi onto seedlings and panicles of rice, Oryza sativa, were evaluated in this study and the efficiency of the inoculation methods assessed by success rates and recovery rates. Among the three methods leading to the infection of young seedlings without obvious mechanical wounds, i.e., water flotation, seed soaking and leaf spraying, the inoculation of 125 nematodes plant−1 by the water flotation method gave rise to the highest success rate and recovery rate, 95.6 and 8.3%, respectively, in growth chamber experiments. When conducted in the plastic house, seed soaking and leaf spraying of 125 nematodes plant−1 resulted in 75.6 and 66.7% success rate, respectively, and 155.7 and 178.1% recovery rates, respectively. The injection or spraying of 2000 nematodes panicle−1 at the booting or flowering stage gave 100% infection.

Distribusi Nematoda Pucuk Putih Padi Aphelenchoides besseyi di Pulau Jawa

Distribution of Rice White Tip Nematode Aphelenchoides besseyi in Java Island Aphelenchoides besseyi is one of the most important rice seed borne nematode causing white tip disease in Indonesia. The status of A. besseyi is considered as quarantine pest category A2, with limited distribution area in Java, Sumatera, and South Kalimantan). The aim of this research was to detect and identify A. besseyi from rice seed based on morphological, morphometry and molecular approaches and to determine its distribution in rice growing areas in Java. Rice seed samples, consisting of 26 varieties, were obtained from seed producers, seed distributors, and farmers in Java. Nematode extraction was done using Baermann method. Molecular identification was carried out by polymerase chain reaction and followed by DNA sequencing and nucleotide analysis. A. besseyi was detected from 74.32% of seed samples. A. besseyi was confirmed to be distributed in Lebak, Bogor, Sukabumi, Subang, Indramayu, Klaten, Sragen, Sukoharjo, Boyolali, Pati, Pemalang, Yogyakarta Sleman, Magetan, Blitar, Tuban, Gresik, Nganjuk, and Banyuwangi. Further nucleotide analysis showed that A. besseyi isolates from rice seed cv Ciherang (Yogyakarta) and cv IR 64 (Banyuwangi) have 99% homology to those from India, China, USA; and 98% homology to those from Taiwan.