Background
Rice (
Oryza sativa) is one of the world’s major cereal food crops. Rice production is seriously threatened by viral diseases [
1], which have become an important problem. As many as 17 rice viruses, widely distributed in rice production areas of Asia and Africa, have been reported. Most of these viruses are transmitted by insects and cause intermittent outbreaks [
2‐
4].
Rice stripe mosaic virus (RSMV) is a tentative new species first detected in southern China in 2015. RMSV is the first reported
Cytorhabdovirus (family
Rhabdoviridae) transmitted by the leafhopper
Recilia dorsalis and the first to infect rice. Previous research has shown that the virus is widely distributed in southern China, where the incidence of infection in some fields exceeds 70% and causes serious rice production losses [
2].
RSMV-infected rice is mainly characterized by slight dwarfing, the presence of twisted leaves exhibiting striped mosaicism, an increased number of tillers, inferior heading and mostly unfilled grains [
2]. Although these symptoms have been reported, previous studies of RSMV have not addressed the viral disease symptom process, symptomatic differences among rice varieties or effects on yield components. In this study, we accordingly investigated RSMV symptoms and their effects on yield factors of representative
indica, hybrid and
japonica rice varieties (cultivars Meixiangzhan, Wuyou 1179 and Nipponbare, respectively) following artificial inoculation in an insect-proof room. Our findings can serve as a basis for disease diagnosis in the field and the formulation of control strategies.
Materials and methods
Rice, virus and vector insect
Three representative rice varieties,
indica rice Meixiangzhan,
japonica rice Nipponbare and hybrid
indica rice Wuyou 1179, were purchased from a commercial supplier (Guangdong South China Agricultural University Seeds, China). Plants infected with RSMV and
R. dorsalis leafhoppers were collected in Luoding, Guangdong Province, China, and maintained in a greenhouse in our laboratory [
5].
Rice culture
Seeds of the tested rice varieties were soaked in water for 12 h, germinated for 2 days at 37 °C, and then sown in organic peat soil (Jiffy). The germinated seedlings were grown in a greenhouse at 28 °C and 60% relative humidity under long-day conditions (16-h light/8-h dark) until the three-leaf stage [
6]. Seedlings at the same growth stage were selected for inoculation with RSMV.
Virus inoculation and detection
Caged, late-stage nymphs of R. dorsalis were allowed to feed on RSMV-infected rice plants at the tillering stage for 16 days. Test seedlings of rice at the three-leaf stage were then inoculated using viruliferous (RSMV) or virus-free (mock) leafhoppers at a ratio of three insects per plant for 3 days. After removal of insects, the inoculated plants were transplanted into a round area with a diameter of 29 cm per plant in an insect-proof room and cultured to maturity at 25 °C − 30 °C and 60–80% relative humidity under long-day conditions (16-h light/8-h dark). The experiment was carried out from April to May 2018, with three biological replicates processed independently. At least 30 plants per rice variety were included in each replicate.
Total RNA was extracted from new leaf tissue (~ 100 mg) taken from the upper part of rice seedlings at 15 dpi using Trizol reagent according to the manufacturer’s instructions (Vazyme Biotech, China). Reverse transcription polymerase chain reaction (RT-PCR) amplification of the viral RNA was carried out using a HiScript II One-Step RNA PCR kit (Dye Plus) P612–01(Vazyme Biotech), and the virus was detected using a procedure similar to a previously reported method [
2]. Virus-positive plants were defined as infected, while virus-negative plants were considered to be healthy. The plants were monitored for symptoms, and yield factors were measured.
Observation of symptoms and measurement of relevant indicators
Symptoms of tested rice plants were observed, plant heights measured and symptomatic rates counted at 5, 10, 15, 20, 25, 30, 45 and 60 dpi. When 100% of healthy control plants had headed, the number of heading virus-infected plants was measured and the heading rate of the infected plants was calculated.
A SPAD-502 Plus chlorophyll meter (Minolta, Osaka, Japan) was used to determine the chlorophyll content of upper, middle and lower portions of tested rice plant leaves at 45 dpi. The average SPAD value of the three leaf parts was used as an indicator of leaf chlorophyll content [
7].
At 45dpi, the middle part of the test plant leaves (~ 5 g) were collected and dried at 65 °C for 48 h. After pulverization, the samples were passed through 40- to 80-mesh sieves and extracted with a 2:1 mixture of acetone and alcohol. After carbonization of cellulose with 72% sulfuric acid, the extract was diluted with distilled water and heated at 121 °C for 1 h. The hydrolysate was filtered, and cellulose, hemicellulose and lignin contents were measured using a 940 Professional IC Vario instrument. Further, the data was converted cellulose and hemicellulose content according to National Renewable Energy laboratory’s standard [
8].
Yield loss determination
At rice plant maturity, the following parameters were measured: the number of tillers, plant height, panicle length, effective panicle number, filled grain number, empty grain number, total grain number, single panicle weight and kernel weight.
Data analysis
The statistical software package SPSS 20.0 (IBM, Armonk, NY, USA) was used to analyze the experimental data. The data were analyzed by one-way analysis of variance (ANOVA) to check for significant differences between infected and healthy plants at P < 0.05 and P < 0.01. Pairwise differences between different cultivars and the significant differences between infected and healthy plants in percentage were evaluated using the nonparametric Kruskal-Walli tests.
Discussion
The three rice varieties infected with RSMV exhibited slight dwarfing, striped mosaicism, stiff, crinkled and even twisted leaves, an increased number of tillers, delayed heading, cluster-shaped shortening of panicles and mostly unfilled grains. Among these symptoms, mosaicism appeared earlier and crinkling later, with both symptoms receding in some infected plants. Yield losses due to RSMV also differed among varieties, with the most serious yield reduction observed in indica cultivar Meixiangzhan, followed by hybrid Wuyou 1179 and then japonica cultivar Nipponbare. Compared with healthy plants, infected varieties had single panicle weights, seed setting rates and 1000-kernel weights that were reduced by 85.42, 94.85 and 31.56% (Meixiangzhan); 52.43, 53.06 and 25.65% (Wuyou 1179); and 25.53, 49.32 and 23.86% (Nipponbare), respectively.
RSMV is a recently identified, tentative new rice virus. Because published studies on the virus are limited, the symptoms of RSMV are still relatively unknown. In the present investigation, we studied the symptoms of RSMV-infected rice by simulating field environmental conditions, with the goal of providing a biological basis for a follow-up theoretical study of RSMV. According to our research, the symptoms of RSMV can be clearly recognized in the field, thus providing a reference for early identification of the disease and timely prevention and control measures according to the disease situation.
Although the leaf stripe mosaic symptom of RSMV-infected plants is similar to that of rice infected with rice stripe virus (RSV, transmitted by the small brown planthopper (SBPH;
Laodelphax striatellus),
Terthron albovittatum,
Unkanodes sapporonus and
Unkanodes albifascia), some characteristic differences exist. At the early infection phase, both viruses cause mottled stripes on leaves of infected rice; at the later infection stage, however, significant differences are observed. In particular, the mosaic symptoms of RSMV gradually diminish or even disappear, whereas mottled leaves infected with RSV whiten or even wither [
9]. In addition, RSV-infected leaves exhibit a drooping phenotype, while RSMV-infected rice has erect leaves that are more rigid than those of healthy plants. This latter characteristic is similar to the symptoms of rice dwarf virus (RDV, transmitted in a persistent manner by
Nephotettix cincticeps,
N. nigropictus,
Recilia dorsalis, and some other
Nephotettix spp.), rice gall dwarf virus (RGDV, transmitted in a persistent manner by
N. cincticeps,
N. nigropictus,
Recilia dorsalis, and some other
Nephotettix spp.), rice black-streaked dwarf virus (RBSDV, transmitted in a persistent manner primarily by the planthopper,
Laodelphax striatellus,
U. sapporonus and
Ribautodelphax albifascia) and southern rice black-streaked dwarf virus (SRBSDV, transmitted by
Sogatella furcifera in a persistent propagative manner) [
3,
5,
6,
10]. Leaf tensile strength and stiffness, which mainly depend on cellulose, hemicellulose and lignin contents, are also related to hormonal changes in plants [
11‐
13]. In our study, cellulose, hemicellulose and lignin contents of leaves of RSMV-infected plants changed significantly. The increased tiller number symptom of RSMV is comparable to that of rice bunchy stunt virus (RBSV, transmitted in a persistent manner by
Nephotettix cincticeps and
N. virescens), RDV and rice grass stunt virus (RGSV, transmitted in a persistent manner by the brown planthopper
Nilaparvata lugens and by two other
Nilaparvata spp.) and differs from that of RGDV and rice yellow stunt virus (RYSV, transmitted in a persistent manner by
Nephotettix cincticeps,
N. nigropictus, and
N. virescens) [
3,
14‐
18]. Similar to the effects of many rice virus diseases, RSMV-infected plants exhibit dwarfing; however, the degree of dwarfing induced by RSMV is far less than that from SRBSDV, RGDV, RBSDV, RDV, rice ragged stunt virus (RRSV, transmitted in a persistent manner by the brown planthopper,
Nilaparvata lugens) and RYSV but is similar to that caused by RSV [
6,
19‐
23].
The mosaicism and crinkling symptoms observed in RSMV-infected leaves may recede, which is similar to the progression of leaf yellowing caused by RYSV [
24,
25]. Both viruses are species in the
Rhabdoviridae family, the former in the genus
Cytorhabdovirus and the latter in
Nucleorhabdovirus. More interestingly, leaf symptoms caused by barely yellow striate mosaic virus (BYSMV; genus
Cytorhabdovirus, family
Rhabdoviridae) also recede [
26,
27]. Whether recession is a common feature of
Rhabdoviridae-caused diseases thus deserves further study [
28‐
30].
Rice virus diseases have the characteristics of burstiness and destructiveness, which cause intermittent disasters in eastern and southeastern Asia. For example, RSV and RBSDV transmitted by the small brown planthopper
Laodelphax striatellus caused serious damage to rice production in the 1960s and 2000s [
3,
10,
31‐
35], and RDV and RYSV transmitted by leafhoppers in the 1980s are widespread in Vietnam, China and Japan [
3]. Since 2009, SRBSDV has rapidly spread and has caused serious rice losses in northern Vietnam and southern China [
36]. RSMV is transmitted by the leafhopper
R. dorsalis in a persistent-propagative manner [
5]. Since its discovery in 2015, the virus has spread rapidly in southern China [
35]. More intense disease monitoring is thus needed. The progression of disease symptoms revealed in our study provide useful information for disease field investigations.
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