Occurrence, distribution, genetic variability and biological properties of bacterial rice pathogens in Cambodia

概要

Rice (Oryza sativa L.) is one of Cambodia's most important field crops, being the staple food for the population, and is also a vital export commodity. Along with this demand, Cambodia produced around 7.9 million tonnes of paddy rice in the wet season. However, a great portion of Cambodian farmers' income is at risk because of possible pest and disease outbreaks. In 2016, Cambodian MAFF reported that rice production is reduced by around 20-30% due to several factors such as pests, climate change, and soil degradation. To control these problems, farmers apply different management measures that include misuse of pesticides because of weak enforcement of current regulations and alternative pest management techniques. As pest management in Cambodia is mainly to promote best practices and is at the developing stage, the current key challenges are to gather practical solutions and to develop strategies to promote this further.

　The yield and quality of paddy rice are affected by biotic and abiotic stresses, and one major threat is bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo). In Asian countries such as China, Vietnam, Indonesia, India, and the Philippines, BLB became one of the most severe and widespread diseases, especially in the irrigated and rained lowland ecosystems. In general, yield loss is around 20 -30% and may sometimes go up to 80% under a severe epidemic. The use of resistant rice varieties is the most promising approach to control the disease. Although the threat of Xoo is recognized, no fundamental information on the disease and pathogen is accumulated in Cambodia. This study aimed to understand Xoo populations and identify the rice resistance genes effective against Xoo isolates in Cambodia. A total of 145 bacterial samples from five provinces were subjected to genotyping and pathotyping. Single nucleotide polymorphism analysis revealed that 53.75% of tested bacteria were known genotypes (population ID 3, 4, 8, 10, 13), but around half of the isolates were unknown populations, suggesting a presence of unique indigenous Xoo genotype(s) in Cambodia.

　Selected 16 bacterial isolates were inoculated to 23 near-isogenic rice lines that carry single (10 plants) or multiple resistant genes (13 plants), and a local variety, Sen Pidor. Based on the lesion length, 16 Xoo isolates were classified into seven pathotypes (A to G) that are further grouped into four by Fisher's Least Significant Difference test: Group 1, pathotypes A and G (12.5%); group 2, athotypes B and F (18.75%); group 3, pathotypes C and E (18.75%); group 4, pathotype D (50%). All Xoo groups were virulent on the rice lines possessing single resistant genes (Xa4, xa5, Xa7, Xa10, Xa11, xa13, Xa14, Xa21, Xa23, and Xa27) with similar lesion lengths on the susceptible variety IR24 (Xa18). However, combinations of resistant genes generally controlled Xoo isolates, if not all, and particularly, Xa4+xa5+Xa21 and Xa4+Xa7+Xa21 resulted in moderate resistant - resistant to all tested Xoo isolates. The local variety showed a moderate level of Xoo-resistance but was susceptible to four bacterial isolates tested. The obtained information can be taken into account for a BLB-resistant rice breeding program in Cambodia.

　The other bacterial disease of rice, rice orange leaf disease, has been reemerged in several Asian countries. The causal pathogen, Rice orange leaf phytoplasma (ROLP), belongs to the “Candidatus Phytoplasma asteris” 16SrI-B subgroup. The ecological and biological characteristics of ROLP are largely unknown because the disease previously had not widely caused serious problems, leading to a low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognized in Thailand, and rice plants showing orange leaf discoloration have become ubiquitous in paddies in the Philippines and Cambodia. Therefore, this study conducted a series of field surveys in two provinces of the Philippines, two provinces of Vietnam, and five provinces of Cambodia. As a result, most suspected ROLP-infecting plants were positive by nested PCR detection; for example, in the total of 98 symptomatic rice plants collected from two provinces of the Philippines, 82% (Laguna) and 95% (Mindanao) were ROLP-positive. These plants showed more varying symptoms than previously reported. A phylogenetic analysis of 16S rRNA genes of ROLP isolates revealed that isolates in Thailand were diverse, and those in India and Cambodia were fallen in the cluster of Thai isolates, suggesting their potential origins. In contrast, those in China, the Philippines, and Vietnam were almost identical and formed an independent group. This result implies a recent expansion event of a single ROLP population into these countries.

　ROLP is solely transmitted by the zigzag-striped leafhopper (Recilia dorsalis Motchulsky) and the green leafhopper (Nephotettix cincticeps Uhler) (Hemiptera Cicadellidae). However, the vector insect R. dorsalis was scarcely present, while N. cincticeps was not observed in the paddies in the Philippines and Cambodia. Instead, green paddy leafhopper, N. virescens Distant (Hemiptera: Cicadellidae), was commonly observed in the paddies throughout all rice growing stages and different cropping seasons. Thus, the ability of N. virescens to transmit ROLP was thoroughly investigated. Newly emerged adult N. virescens, which fed on ROLD-source rice plants, were used to inoculate a susceptible rice seedling and were serially transferred into a new healthy seedling. The resultant positive transmission rates varied from 5.1% to 17.8%. The transmission ability of the insects was generally decreased over time. These findings suggest that N. virescens is an alternative vector of ROLP in the Philippines and Cambodia.

　The ROLP-infected rice exhibits clear orange to yellowish leaf discoloration and severe stunting in seedlings, resembling symptoms caused by rice tungro viruses. Serological and genetic detection of tungro viruses suggested mostly no virus infection in the tested rice plants. Moreover, fluorescent and scanning electron microscopic observations revealed the intensive accumulations of the phytoplasma in phloem tissues. The massive accumulation of storage starch in vascular bundle sheath and parenchyma suggested a correlation between the symptoms and the plant’s energy conversion defectiveness. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen’s biological impact on rice tissue. Moreover, the study highlighted the increasing importance of ROLD-reemergence in Southeast and East Asia. It proved the need to manage R. dorsalis, N. virescens, and N. cincticeps carefully.