Feeding selectivity of Aphelenchoides besseyi and A. pseudogoodeyi on fungi associated with Florida strawberry

Aphelenchoides besseyi and A. pseudogoodeyi are foliar nematodes associated with commercial strawberry production in Florida, USA. The reproductive and feeding habits of these two nematode species were assessed on Florida isolates of the fungi Botrytis cinerea, Colletotrichum gloeosporioides, Macrophomina phaseolina, and Neopestalotiopsis spp. pathogenic to strawberry, and the non-pathogenic isolates of Fusarium oxysporum and Monilinia fructicola grown on PDA in petri dishes. Each culture was inoculated with six specimens with mix life stages of either A. besseyi and A. pseudogoodeyi and incubated at 24°C under axenic and non-axenic conditions 23 and 31 days after inoculation, respectively. Aphelenchoides besseyi reproduction rates were significantly higher on strawberry pathogenic isolates of B. cinerea, C. gloeosporioides, and Neopestalotiopsis rosae than on the non-pathogenic isolates of F. oxysporum and M. fructicola. In contrast, reproductive rates of A. pseudogoodeyi did not significantly vary across the fungi cultures. For both nematode species, Macrophomina phaseolina was a poor host because it did not produce mycelium on the media used. Our findings indicate that A. besseyi is more selective in its fungal-feeding preference than A. pseudogoodeyi. Additionally, A. pseudogoodeyi eggs and juveniles were significantly higher than adults. Yet, for A. besseyi, adult stages were more common. Fungi aid in the maintenance of soil-dwelling populations of these two nematode species. Removing fungi-infected strawberry plant residues is both a desirable and effective management practice to limit A. besseyi in central Florida commercial strawberry fields.

Phytonematodes as a limiting biotic factor of agricultural production in the world (review)

Purpose. To analyze domestic and foreign scientific literature on the species composition and harmfulness of the world's most dangerous parasitic species of phytonematodes in crops. Results. Today, according to the available literature, the most dangerous species of phytonematodes include: gall nematode (Meloidogyne spp.), cyst-forming nematode (Heterodera spp. and Globodera spp.), root lesion nematode (Pratylenchus spp.), banana drill nematode (Radoholus similis), stem nematode (Ditylenchus dipsaci), pine stem nematode (Bursaphelenchus xylophilus), reniform nematode (Rotylenchulus reniformis), xiphinema index (Xiphinema index), false head nematode (Nacobbus aberrans), and rice leaf nematode (Aphelenchoides besseyi). Conclusions. The results of research on the prevalence and harmfulness of parasitic nematode species in crops convince us of the need for a more detailed study of this group of microorganisms. Due to the rapid development of molecular genetic methods in the last decade, scientists have been able to expand and improve their knowledge of identifying species, races and pathotypes of phytonematodes, their biological and environmental characteristics, and most importantly, to discover and understand extremely complex mechanisms of parasite and host plants. Nematologists are confident that further research in these and other areas in the future will create a basis for developing a new strategy for long-term and environmentally safe control of these dangerous plant parasites.

Aphelenchoides besseyi parasitizing cowpea (Vigna unguiculata) in Brazil

Several species of nematodes are known to cause losses to cowpea (Vigna unguiculata) throughout the world. In Brazil, Aphelenchoides besseyi was recently described causing damages on soybean, cotton, and common bean, but no report was found about the parasitism of this nematode in cowpea. The present study aimed to verify the host reaction of cowpea cultivars to A. besseyi. The experiment was conducted under greenhouse conditions, using as inoculum two A. besseyi populations, obtained from symptomatic soybean and cotton plants collected in naturally infested fields. Cultivars ‘Imponente’, ‘Aracê’, ‘Guariba’, ‘Tumucumaque’, ‘Nova Era’, and ‘Tracuateua’ were inoculated with 500 A. besseyi of each population, separately, into soil and after 30 days from the inoculation nematodes were extracted from shoot systems. Both populations were able to parasitize all the cowpea cultivars. Independently of the cultivar, cowpea plants exhibited symptoms of leaf deformation similar to those described for soybean, cotton, and common bean and, in addition, severe brooming was observed and the interior of the stems was porous and necrotic. To our knowledge, this is the first report of parasitism by A. besseyi of cowpea in Brazil, under greenhouse conditions, increasing the list of hosts of this nematode.

Long-term population dynamics of Aphelenchoides besseyi on Oryza sativa in a paddy field, and changes in the pathological and ecological traits of the two populations

Summary An Oryza sativa cultivar inoculated with the white tip nematode, Aphelenchoides besseyi, was cultivated over 18 years to investigate the population dynamics of the nematode and the change in the virulence of nematodes and tolerance/resistance of plants. The mean number of living nematodes per seed (S) showed two peaks, each of which was followed by a 3-year decrease in the S value, during the initial 10 years. The annual reproduction curve between in year and in year t had a peak in a range of of <1.1. The incidence of white tip disease increased with increasing mean number of living nematodes seed−1 in the previous year, whereas the proportion of seeds with living nematodes increased with increasing mean number of living nematodes seed−1 in the current year. Inoculation tests of the nematode cohorts on the seedling cohorts derived from seeds harvested in the same or different years indicated that the initial 6-year interaction between the rice and nematode populations caused the nematodes to increase the incidence of white tip disease and to decrease the degree of seed swelling and the proportion of heavy seeds. On the other hand, the interaction caused the rice plants to prevent the incidence of disease from increasing and the degree of seed swelling and the proportion of heavy seeds from decreasing.

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.

A novel ATPase gene, Ab-atps, plays an important role in the interaction of rice and white tip nematode, Aphelenchoides besseyi

Plant kinases containing the LysM domain play important roles in pathogen recognition and self-defense reactions. And it could recognize microbe-associated molecules including chitin and other polypeptides. The white tip nematode Aphelenchoides besseyi is a migratory parasitic nematode that infects plant shoots. It is distributed over almost all rice-producing areas and causes up to 50% economic losses. The rice OsRLK3 gene was a defense-related LysM kinase gene of rice. This study showed that the rice LysM kinase OsRLK3 could be induced by flg22, jasmonic acid, salicylic acid, and chitin. An interaction gene, Ab-atps from A. besseyi, was identified by screening the interaction between the rice gene OsRLK3 and an A. besseyi cDNA library using yeast two-hybrid screening. Ab-atps is a novel ATP synthase gene with a full length of 1341 bp, coding for 183 amino acids. The mRNA of Ab-atps was located in the esophagus and reproductive system of A. besseyi. The expression of Ab-atps was assessed at different developmental stages of the nematode and found to be the highest in the juvenile, followed by the egg, female, and male. Reproduction was significantly decreased in nematodes treated with Ab-atps double-stranded RNA (dsRNA) (p < 0.05). Transient expression experiments showed that Ab-ATPS-GFP was distributed in the nucleus, cytoplasm, and cell membrane, and Ab-ATPS-GFP triggered plant cell death. OsRLK3 was expressed significantly higher at 0.5 day and 1 day (p < 0.05) in rice plants inoculated with nematodes treated with Ab-atps dsRNA and gfp dsRNA for 0.5–7 days, respectively. Further, OsRLK3 expression under Ab-atps dsRNA treatment was significantly lower than with gfp dsRNA treatment at 0.5 day (p < 0.05) and significantly higher than with gfp dsRNA treatment at 1 day (p < 0.05). These results suggest that rice OsRLK3 could interact with A. besseyi Ab-atps, which plays an important role in growth, reproduction, and infection of the nematode. Our findings provide a theoretical basis to further understand the parasitic strategy of A. besseyi and its interaction mechanism with host plants, suggesting new ideas and targets for controlling A. besseyi.

Molecular Characters of AB-FAR Gene 1 of Aphelenchoides besseyi from Five Rice Varieties

Aphelenchoides besseyi merupakan nematoda penyebab penyakit pucuk putih yang terbawa benih padi. Gen AB FAR-1 diketahui sebagai gen penting yang mengendalikan patogenisitas A. besseyi. Penelitian dilakukan untuk mengetahui karakter gen AB FAR-1 yang diisolasi dari nematoda yang berasal dari benih padi. Ekstraksi nematoda dilakukan dengan metode corong Baerman dari benih 5 varietas padi “Ciherang“, “Inpari Sidenuk“, “Sintanur“, “Hibrida Prima“ dan “Pak Tiwi“. Ekstraksi DNA total nematoda menggunakan metode CTAB dilanjutkan dengan amplifikasi gen AB FAR-1 menggunakan primer spesifik FAR-F1/R1 dan analisis urutan nukleotidanya. Pita DNA spesifik gen AB FAR-1 berukuran 150 pb berhasil diamplifikasi dari semua sampel nematoda. Analisis sekuen menunjukkan bahwa gen AB FAR-1 tersebut memiliki homologi tertinggi (92.5 – 100%) dengan aksesi Genbank JQ686690.1, yaitu gen AB FAR-1 A. besseyi asal Cina. Walaupun memiliki homologi yang tinggi, terdapat beberapa perbedaan nukleotida pada sampel gen AB FAR-1 A. besseyi asal “Ciherang“, “Inpari Sidenuk“ dan “Hibrida Prima“. Analisis pohon filogenetika lebih lanjut mengelompokkan gen AB FAR-1 A. besseyi menjadi 2 grup, yaitu grup 1 terdiri atas gen AB FAR-1 A. besseyi asal Cina, “Sintanur“, “Hibrida Prima“ dan “Pak Tiwi“ dan grup 2 gen AB FAR-1 A. besseyi asal “Ciherang“, dan “Inpari Sidenuk“.

The ACE Genes in Aphelenchoides Besseyi Isolates and Their Expression Correlation to the Fenamiphos Treatment

Aphelenchoides besseyi could cause great yield loss on rice and many economically important crops. Acetylcholinesterase inhibitors were commonly used to mitigate plant parasitic nematodes. However, increasing nematicide-resistance has been reported due to the extensive use of these chemicals. The correlation between the AChE-inhibitor (fenamiphos) sensitivities and acetylcholinesterase (ace) genes in two isolates of A. besseyi (designated Rl and HSF) was established. The LD50 of fenamiphos to Rl and HSF were 572.2 ppm and 129.4 ppm, respectively, indicating that two nematode isolates had different sensitivities to fenamiphos. Three ace genes were cloned and sequenced in A. besseyi, and their homology was supported by phylogenic analysis with AChEs protein sequences from various vertebrate and invertebrate species. Molecular docking showed that the affinities of each AChEs to fenamiphos were higher in HSF isolate, indicating that there should be point mutations in Rl isolate AChEs. Treating the two isolates with 100 ppm fenamiphos for 12 h, three ace genes of HSF isolate were down-regulated but were up-regulated in Rl isolate. The results suggest that fenamiphos can transcriptionally modulate the expression of ace genes, as well as the variants in AChEs and increased expression of ace genes might be associated with fenamiphos-insensitivity in Rl isolate.

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.