Symptoms and yield loss caused by rice stripe mosaic virus

Rice stripe mosaic virus (RSMV) is a tentative new Cytorhabdovirus species in family Rhabdoviridae transmitted by the leafhopper Recilia dorsalis. Although the virus was first detected in southern China in 2015, few studies have investigated rice symptoms and yield losses caused by RSMV infection. In this study, we observed and systematically compared symptoms of three virally infected, representative varieties of indica, hybrid and japonica rice and determined the yield parameters of the artificially inoculated plants. The three RSMV-infected cultivated rice varieties exhibited slight dwarfing, striped mosaicism, stiff, crinkled or even twisted leaves, an increased number of tillers, delayed heading, cluster-shaped shortening of panicles and mostly unfilled grains. Slight differences in symptom occurrence time were observed under different environmental conditions. For example, mosaic symptoms appeared earlier and crinkling symptoms appeared later, with both symptoms later receding in some infected plants. Yield losses due to RSMV also differed among varieties. The most serious yield reduction was experienced by indica rice (cv. Meixiangzhan), followed by hybrid indica rice (cv. Wuyou 1179) and then japonica (cv. Nipponbare). Single panicle weight, seed setting rate and 1000-kernel weight were reduced in the three infected varieties compared with healthy plants—by 85.42, 94.85 and 31.56% in Meixiangzhan; 52.43, 53.06 and 25.65% in Wuyou 1179 and 25.53, 49.32 and 23.86% in Nipponbare, respectively. Our findings contribute basic data for field investigations, formulation of prevention and control strategies and further study of the pathogenesis of RSMV.


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][3][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.

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]. Viruspositive plants were defined as infected, while virusnegative 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 virusinfected 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 80mesh 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.

Characteristic symptoms
In our three experiments, the RSMV-infected Meixiangzhan plants that we obtained were respectively 25(83. Typical RSMV symptoms of plant dwarfing and striped or spotted, stiff, crinkled or even twisted leaves were observed in the three tested varieties (Fig. 1a-c). No  (Fig. 1d). The three rice varieties were dwarfed to different extents after RSMV infection: Meixiangzhan exhibited the highest degree of dwarfing, followed by Nipponbare and then Wuyou 1179 (Additional file 1: Figure S1).
Leaf chlorophyll contents, as reflected by SPAD values, of the infected varieties Meixiangzhan, Nipponbare and Wuyou 1179 were reduced respectively by 20.71, 33.86 and 19.40% of those of healthy control plants at 45 dpi, thus indicating that the chlorophyll contents of infected plants were obviously decreased (Fig. 1e). The degree of reduction differed significantly among the three varieties; the highest reduction in SPAD values was in Nipponbare, followed by Meixiangzhan and then Wuyou 1179 (Additional file 2: Figure S2).
Cellulose, hemicellulose and lignin contents of plant leaves affect leaf morphology. Infection with RSMV can increase the stiffness of rice leaves, a change that is readily apparent by manual comparison of infected and healthy leaves. At 45 dpi, lignin contents of the three tested varieties were increased (Fig. 1f). Cellulose contents of Meixiangzhan and Wuyou 1179 were decreased, and the hemicellulose contents of three varieties were also decreased (Additional file 15: Table  S1). Glucose contents of indica and hybrid rice varieties decreased by 9.32 and 6.87%, respectively (Additional file 3: Figure S3). Galactose contents of the three tested varieties (Meixiangzhan, Nipponbare and Wuyou 1179) increased by 5.69, 0.24 and 5.01%, respectively (Fig. 1g). Changes in the contents of these substances may be responsible for the stiffening of leaves.
The process of characteristic symptom development The first disease symptom observed on RSMV-infected plants was mosaic stripes on leaves, which generally appeared at the tip of newly emerged leaves. In three repeated trials at 10 dpi, the incidence of mosaicism on the three cultivars was as follows: indica rice Meixiangzhan, 81.82, 43.75 and 5.56%; japonica rice Nipponbare, 56.52, 13.64 and 22.22%; and hybrid rice Wuyou 1179, 13.04, 30.00 and 3.85% (Fig. 2a). At 15 dpi, the incidence of mosaicism on the three tested varieties had increased significantly, to more than 80%. In the worst case, 90.91% of Meixiangzhan plants were affected. The mosaic symptoms of some infected plants later receded (Fig. 2a). As the disease developed, the proportion of mosaic leaves increased first and then decreased (Additional file 4: Figure S4). SPAD values, representing chlorophyll content, were always lower in infected plants than in healthy ones; the largest differences between infected and healthy plants were observed from 5 to 10 dpi and 30 to 45 dpi, and the smallest difference was from 15 to 25 dpi (Additional file 5: Figure S5).
Dwarfing was first observed at 15 dpi. By the end of the experiment, infected plants of the three tested varieties were approximately 80% of healthy control plants (Additional file 6: Figure S6). With the development of the disease, the dwarfing phenotype became increasingly obvious, especially in Wuyou 1179. At 25 dpi, the heights of infected plants were less than 80% of healthy control plants. Dwarfing was most pronounced in Meixiangzhan and Nipponbare during later disease stages. At 45 dpi, heights of Meixiangzhan and Nipponbare were respectively 88.04 and 79.53% of those of healthy control plants (Additional file 6: Figure S6).
Leaf crinkling was observed from 15 to 20 dpi in some infected plants. The emergence time of this symptom varied slightly among varieties, appearing first in Nipponbare, secondly in Meixiangzhan and finally in Wuyou 1179 (Figs. 2b and Additional file 7: Figure S7). At 20 dpi, the incidence of leaf crinkling based on three replicates was 34.78, 18.75 and 11.11% in Meixiangzhan, 13.04, 13.64 and 11.11% in Nipponbare, and 21.74%. 15.00 and 7.69% in Wuyou 1179 (Fig. 2b). At 45 dpi, the incidence of this symptom was significantly increased, reaching more than 60%, with that of Nipponbare and Wuyou 1179 at or above 90% (95.45 and 90.00%, respectively) (Fig. 2b). After an initial increase, the proportion of crinkled leaves per plant declined and then increased. As the disease developed, some leaf crinkling symptoms also receded (Additional file 8: Figure S8).

Symptoms associated with rice yield and yield losses
The number of tillers in the three RSMV-infected varieties increased significantly: by 40.00% in Meixiangzhan, 47.50% in Nipponbare and 55.56% in Wuyou 1179 (Figs. 3a-b and Additional file 9: Figure S9). Among the three varieties, these differences were not significant. In infected plants, heading time was significantly delayed, and heading was incomplete (Fig. 3c). When 100% of healthy control plants had undergone heading, the heading incidence of Meixiangzhan was 42.86% (Experiment II) and 72.73% (Experiment III); that of Nipponbare was only 84.38% (II) and 58.33% (III), while that of Wuyou 1179 was 34.78% (II) and 5.26% (III) ( Table 1).
As the number of tillers increased, the number of rice panicles in RSMV-infected plants also increased. Average numbers of panicles per plant in the three tested varieties were 150.57% (Meixiangzhan), 127.45% (Nipponbare) and 162.29% (Wuyou 1179) of those of healthy plants (Figs. 4b and Additional file 10: Figure S10). In contrast to the increase in panicle numbers, panicle lengths of infected plants were significantly decreased, being only 65.47% (Meixiangzhan), 78.36% (Nipponbare) and 59.08% (Wuyou 1179) of those of healthy ones (Additional file 11: Figure S11). In addition, the panicles of Meixiangzhan and Wuyou 1179 were arranged in short clusters (Fig. 4a).

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 RSMVinfected 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, RSVinfected 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 blackstreaked 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][12][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][15][16][17][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][20][21][22][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][29][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][32][33][34][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.

Conclusions
RSMV-infected plants exhibited slight dwarfing, striped mosaicism, stiff, crinkled or 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 later receding in some infected plants. Yield losses due to RSMV also differed among varieties. Indica rice (cv. Meixiangzhan) experienced the most drastic yield reduction, followed by hybrid indica rice (cv. Wuyou