Field effect of Cnaphalocrocis medinalis granulovirus (CnmeGV) on the pest of rice leaffolder

Journal of Integrative Agriculture 2019, 18(9): 2115–2122 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Field effect of Cnaphalocrocis medinalis granulovirus (CnmeGV) on the pest of rice leaffolder XU Jian, LIU Qin, LI Chuan-ming, HAN Guang-jie Jiangsu Lixiahe Institute of Agricultural Sciences, Yangzhou 225007, P.R.China

Abstract Rice leaffolder, Cnaphalocrocis medinalis (Guenée), has become a major pest throughout the rice cultivating areas of China and caused severe damage to rice production. Cnaphalocrocis medinalis granulovirus (CnmeGV), a naturally occurring baculovirus, is revealed as a potential microbial agent for the pest control. Field applications of CnmeGV were conducted against rice leaffolder larvae in rice paddies. CnmeGV infected the larvae not only in the current generation but also in the successive generation, resulting in a sustained infection in the larva population for at least 48 days. Under diferent concentrations of CnmeGV (7.5×1011 and 1.125×1012 occlusion body (OB) ha–1) at 30 days after spraying, larval population reduced up to 76.32% and rice leaf rolled rate kept in 15.42%. Simultaneously, CnmeGV had no impact on arthropod predators of C. medinalis, with abundances ranging from 2.39 to 3.79 per ten hills. These results revealed that CnmeGV is suitable as a bio-pesticide for rice leaffolder management in rice paddies. Keywords: rice leaffolder, Cnaphalocrocis medinalis granulovirus, field effect

1. Introduction Rice (Oryza sativa L.) is one of the most important crops grown worldwide. Rice is used as primary food source by more than half of the world’s population (Zhang et al. 2016). China has a long history of rice cultivation and is the largest rice producer and consumer in the world. Rice is grown on approximately 20% of the arable land in China, which is larger than that of other agricultural crops (Chen et al. 2011).

Received 17 May, 2018 Accepted 20 August, 2018 Correspondence XU Jian, Tel: +86-514-87637599, E-mail: [email protected] © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(18)62097-0

The rice leaffolder, Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae), is a migratory insect pest of rice in Asia (Dale 1994). The larvae roll the rice leaf longitudinally for shelter, scrape the leaf tissues within the shelter, and thus reduce the efficiency of photosynthesis, which results in a decrease of rice yield up to 30%, depending on population levels and weather conditions (Litsinger et al. 2006; Rani et al. 2007). In recent years, outbreaks of this insect have occurred throughout the rice cultivating areas in China, and have severely threatened rice production (Xu et al. 2011; Liu et al. 2012). Due to the severity of the insect pest infestation, the application of chemical insecticides has mostly been relied on to control the damage. Such pest management practices are increasingly criticized as being incompatible with environmental goals, which aim at developing sustainable agricultural practices and promoting ecosystem services. Baculoviruses are well-known group of insect viruses (Cory and Franklin 2012). The family Baculoviridae has

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now been subdivided into four genera, which comprises Alphabaculovirus (nucleopolyhedrovirus (NPV), infecting lepidopteran hosts), Betabaculovirus (granulovirus (GV), infecting lepidopteran hosts), Deltabaculovirus (dipteran NPV), and Gammabaculovirus (hymenopteran NPV) (Jehle et al. 2006). Most baculoviruses are isolated from infected lepidopteran insects. Some species have been applied for pest control of agriculture and forest successfully (Szewczyk et al. 2006). An important factor contributing to the success of baculoviruses as biological control agents against insect pests is the ability of the virus to persist in the environment and transmit in the populations (Bonning and Nusawardani, 2007). Furthermore, these viruses, which might be a viable alternative to pesticide dependence, may even lead to reducing dependence on pesticides and enhancing agricultural production methods (Elderd 2013). Many researchers have focused on isolating pathogens against rice leaffolder to develop biological control (Kirubakaran et al. 2014). Cnaphalocrocis medinalis granulovirus (CnmeGV) was first isolated from larva of rice leaffolder in 1979 in China (Pang et al. 1981). After more than 30 years, the pathogen was still obtained at the same place where the virus was first isolated, and revealed stable existence in the field and potential effect to the target pest (Zhang et al. 2014). Occasionally, the strain of CnmeGV was isolated from the larvae of rice leaffolder in the paddy of Yangzhou, China (Xu et al. 2016). Bioassay tests indicated that this granulovirus was highly virulent to rice leaffolder larvae (Liu et al. 2013). Recently, the complete sequence and morphological characterization of this virus genome were presented (Han et al. 2016). However, field studies evaluating its efficacy, as well as its economic benefits to farmers are yet to be conducted. In this study, we examined two important parameters of CnmeGV: field control efficacy and viral transmission. The potential of CnmeGV as a biocontrol agent against C. medinalis was discussed.

2. Materials and methods 2.1. Multiplication of GV A colony of C. medinalis was reared in the laboratory, with the improved artificial diet described by Xu et al. (2012). CnmeGV, isolated from Yangzhou and preserved in laboratory, was used to infect the larvae and multiply the occlusion body (OB) of the granulovirus (Liu et al. 2013). The second instar larvae were fed with an artificial diet, incorporated within CnmeGV at a concentration of 105 OB g–1. After 48 h, uneaten food was replaced with untreated and fresh artificial food. Larvae were reared at (25±1)°C for 15 days and individuals showing obvious symptoms of GV infection were collected and homogenized in distilled

water. After filtered through a four-layered cheesecloth and centrifuged at a low speed (1 000×g) for 5 min, debris of larval tissue in the suspension were removed. The supernatant was centrifuged at a high speed (10 000×g) for 15 min, and the deposited granulovirus OBs were washed several times with distilled water. Then, the pellets were re-suspended in distilled water, and the concentration was quantified to 1010 OB mL–1 by dark field microscopy using 0.02-mm depth cytometers.

2.2. Field treatments The study was carried out at Changxing, Zhejiang, China (30°43´N, 119°33´E) during the summer season. The mean annual rainfall was 133 mm, with mean maximum and minimum temperatures of 29 and 21°C, respectively. The area was a typical wheat-rice growth model. The first generation of rice leaffolder usually immigrated at the end of June, and generated the second and third generations, which were the main cause of rice infestation annually. Natural baculovirus infection of the pest was never observed in this place. Testing field was about 0.3 ha and located in tracts of paddy fields. The variety of rice was Xiushui 134 (bred by Agricultural Research Institute of Jiaxing, Zhejiang, China). Applications of CnmeGV were made at the jointing stage of rice and the beginning of egg hatching of the second pest generation. A 15-L knapsack sprayer was used to conduct the various treatments, and the sprayer was thoroughly cleaned with soap and water prior to being re-filled with another formulation for application. Two different concentrations of CnmeGV (7.5×1011 OB –1 ha and 1.125×1012 OB ha–1) were used to determine potential concentration effects. Tween-80 (0.1%) was added to enhance the distribution on the surface of leaves. The negative control in the trial consisted of water and Tween-80 (0.1%). The synthetic pesticide, 40% chlorpyrifos (Dow AgroSciences, China), which was applied at the dose of 1 200 mL ha–1 according to the manufactures’ instruction, was set as the positive control in the trial. The experimental layout was a completely randomized design. All the four treatments were replicated three times within the same field. Each plot was 150 m2 (15 m×10 m) with about 4 500 hills of rice seedlings transplanted and separated by a distance of 1 m with four lines of rice planted.

2.3. Sampling for investigation Damage caused by leaffolder and the number of larvae or pupae of survived C. medinalis were investigated at 6-, 12-, 18-, 24-, 30-, 36-, 42- and 48-day intervals after CnmeGV spraying. Fifty rice hills in each treatment plot were sampled randomly for checking at every investigating

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the means between the treatments were determined using Fisher’s protected least significant difference (PLSD) test. Differences between means were considered significant at P≤0.05. All analyses were conducted using the data processing system (DPS) (Tang and Feng 2002).

time. Rolled leaves and larval survival were recorded respectively. Among the survival samples, larvae presenting symptoms of white body and swollen somites were regarded as virus infected, and were verified by PCR to amplify the conserved gene granulin of granulovirus at the same time (granF: 5´-TGCAACTGGAGCGGAAAAG-3´; granR: 5´-CAATGGGAACGGGAAACCT-3´). Pupae were collected and reared in the laboratory. All eclosed pupae were recorded as survivors. At the peak of moth emergence, about 30 days after spraying, adult abundance of C. medinalis was inspected by counting the number of moths flying around when rice leaves were disturbed. Each plot was flapped with a 1.5-m-long stick for a successive 10-m distance. The abundance of arthropod predators of C. medinalis was determined by counting the total number of spiders and lady beetles presented (adults and larvae), by sweepnet sampling, during the experiment. Five samples were taken per plot, with each sample of five sweeps from 25 hills.

3. Results 3.1. Dynamics of CnmeGV infection CnmeGV-infected larvae showed distinct symptoms with their color changing from dull green to milky-white and somites swelling. Emulsion flowed from the larval body easily when larvae were prodded at late stage. Results of viral infection identified from symptoms were completely in agreement with the transcript level of granulin, a granulovirus conserved gene. CnmeGV infected the larvae of rice leaffolder not only in the current generation, but also in the next generation, resulting in a sustained infection of the larval population in the rice field for at least 48 days. Infection rates fluctuated in the two successive generations, with two obvious infection peaks (Fig. 1). About 6 days after the virus spraying, few larvae showed obvious symptoms of CnmeGV infection, ranging from 6.59 to 11.04% in different treatments. After 12 days, the number of infected larvae increased remarkably; about half of the investigated larvae showed symptoms of infection, and a broad larval infection peak appeared at about 24 days, with an infection rate ranging from 74.72 to 81.38% at different concentrations. About 30 days after the pupation, eclosion, and oviposition of the first larval generation, and the emergence of the next generation, larval infection rates decreased to 32.27–41.9% in different treatments. Another infection peak was observed around 42 days post-spraying, and field infection rates remained around 51–61.41%

2.4. Data analysis The rate of rolled leaf was calculated by comparing the rolled leaves with the total sampled leaves to evaluate the infestation caused by C. medinalis. Larva reduction rate was estimated by comparing the residual larvae of CnmeGV/pesticide treatments with that of negative control to evaluate the effect of CnmeGV on larval mortality. Larva infection rate was the counts of infection larvae divided by the total survival larvae within the treatment. Differences among treatments of leaf rolled, larval infection, larval mortality and predator abundance were assessed by two-way analysis of variance (ANOVA). To identify the trends, further comparisons were made for each sample date using univariate ANOVA. Adult abundance was compared using one-way ANOVA. Multiple comparisons of 90

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Fig. 1 Dynamics of larval infection after spraying with various concentrations of Cnaphalocrocis medinalis granulovirus (CnmeGV) at different stages. OB, occlusion body. Data are mean±SE (n=3). Different letters indicate that means are significantly different (P<0.05).

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in different treatments. Infection rates declined after 42 days due to the insect turning into pupal stage gradually. Except at sampling date of 12 days, no significant difference was observed between different concentrations in the infection rate over the duration of experiment (F1, 32=4.8, P=0.008).

3.2. Pest infestation and larvae mortality Infestation of leaffolder in different treatments in the two successive generations was shown in Fig. 2. It was found that different concentrations of CnmeGV and chlorpyrifos had great effect on the infestation of the pest. Multivariate tests showed significant difference between treatments and the sampling time interaction of leaf rolled rate (F21, 64= 8.9, P=0.0001). Different doses of CnmeGV showed no difference in leaf rolled rate, but were significantly different from the control and chlorpyrifos (F3, 64=276.2, P=0.0001). Leaf rolled rate increased in all the treatments over the duration of the experiment (F7, 64=223.3, P=0.0001). The treatments of CnmeGV had a moderate effect on the infestation of the pest at the early stage, with leaf rolled rates ranging from 2.03 to 2.19% at 6 days post spraying. At 30 days (the late stage of current pest generation), the leaf rolled rates of different CnmeGV treatments were 14.75 and 15.42%, which were significantly different from that of the control (26.97%) (F3, 8=37.51, P=0.0001). The synthetic pesticide, chlorpyrifos, was superior in reducing the number of rolled leaves of C. medinalis in comparison to the other treatments, especially at the early stage (6 days; F3, 8=17.6, P=0.001). In the next pest generation, CnmeGV maintained low leaf Control

rolled rates, ranging from 23.95 to 24.81% at 48 days post spraying, with no remarkable difference from that of chlorpyrifos treatment, but significantly lower than that of the control (F3, 8=45.6, P=0.001). Larval mortalities were also influenced by different concentrations of CnmeGV and chlorpyrifos (Fig. 3). Multivariate tests showed significant difference between treatments and the sampling date interaction of overall larval mortality (F14, 48=35.1, P=0.0001). Effect between different concentrations of CnmeGV was not significantly different, but lower than that of chlorpyrifos (F2, 48=29.3, P=0.0001). In contrast to chlorpyrifos, larval population reduction rates of CnmeGV treatments were significantly lower in sampling time of 6, 12 and 18 days, but remarkably higher in the stage of 36, 42 and 48 days (F7, 48=56.9, P=0.0001). The highest larval mortality of CnmeGV treatments was found at the sampling time of 30 days. Larval population in the treatments of different concentrations of CnmeGV reduced by 76.32 and 80.25%, respectively, but not different to that of chlorpyrifos (F2, 6=4.1, P=0.08). These results indicated that viral infection was progressive and caused the larval mortality dynamically over the duration of the experiment. In the following generations, CnmeGV still maintained a sustainable infection on the larvae, causing a mortality rate of up to 31.54% at 48 days, while the chlorpyrifos treatment kept larval mortality rate at only 8.25% (F2, 6=10.2, P=0.01).

3.3. Abundance of C. medinalis adults and arthropod predators Adult density of C. medinalis was high in the control at 30

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Fig. 2 The leaf rolled rates of rice in different treatments at different stages. Control, treated with water and Tween-80 (0.1%); chlorpyrifos, 40% chlorpyrifos at dose of 1 200 mL ha–1; CnmeGV, Cnaphalocrocis medinalis granulovirus; OB, occlusion body. Data are mean±SE (n=3). Different letters indicate significant difference (P<0.05).

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days after treating, while average adult densities of all the other treatments were significant low (F3, 8=161.5, P=0.001) (Fig. 4). CnmeGV greatly suppressed the population of adult C. medinalis, and the adult densities ranged from 0.3 to 0.39 moths m–2 in plots treated with different concentrations, which were about 79.05 to 83.23% less than that of the control. High concentration of CnmeGV showed relative high effect. However, there was no remarkable difference between different concentrations (F1, 4=0.61, P=0.478). Compared to the positive control, the moth densities in the CnmeGV treatments were similar to that in the synthetic pesticide, with no significant statistical difference (F2, 6=2.425, P=0.169). Abundance of arthropod predators of different treatments following the sampling time was shown in Table 1. Statistical analysis indicated no significant difference between treatments by time interaction during the study period (F21, 64=0.61, P=0.156). The abundance of different concentrations of CnmeGV had no obvious difference from that of the control, but significantly differed from the treatment of chlorpyrifos at any sampling date (F3, 64=90.01, P=0.0001). CnmeGV had no impact on the arthropod predators of C. medinalis. During the study period, high densities of arthropod predators were observed in CnmeGV treatments, with numbers ranging from 2.39 to 3.79 per ten hills. The synthetic pesticide greatly affected the population of the arthropod predators of rice leaffolder, especially at the early stage after spraying. The density of predators was only 0.01 per ten hills (6 days; F3, 8=21.35; P=0.001). Though the density increased gradually at late stage, to 2.35 per

Moth density (no. m–2)

Fig. 3 Larval mortality of Cnaphalocrocis medinalis in different treatments at different stages. Chlorpyrifos, 40% chlorpyrifos at dose of 1 200 mL ha–1; CnmeGV, C. medinalis granulovirus; OB, occlusion body. Data are mean±SE (n=3). Different letters indicate significant difference (P<0.05).

Fig. 4 Adult abundance of Cnaphalocrocis medinalis in the paddy at the stage of 30 days after different treatments. CnmeGV, C. medinalis granulovirus; OB, occlusion body; chlorpyrifos, 40% chlorpyrifos at dose of 1 200 mL ha–1; control, treated with water and Tween-80 (0.1%). Data are mean±SE (n=3). Different letters indicate significant difference (P<0.05).

ten hills on day 48, this was still lower than that of CnmeGV treatments and control (F3, 8=6.87; P=0.013).

4. Discussion Baculoviral diseases of insects are characterized by a rapid multiplication in the tissues of hosts, persistence in the environment, and the ability to prevail within host populations. Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts (Dwyer et al. 2000). Most epizootiological studies focused

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Table 1 Abundance of arthropod predators in different treatments at different stages in the paddy Treatment1) CnmeGV 7.5×1011 OB ha–1 CnmeGV 1.125×1012 OB ha–1 Chlorpyrifos Control

6d 2.93±0.12 2.77±0.72 0.01±0.01** 2.57±0.74

12 d 3.30±0.82 3.13±0.32 0.10±0.06** 3.25±0.39

Abundance (no. per ten hills) 18 d 24 d 30 d 36 d 2.72±0.20 3.39±0.39 2.39±0.76 2.84±0.88 2.58±0.72 3.33±0.77 2.54±0.56 3.07±0.55 0.61±0.36** 0.69±0.54** 0.64±0.32** 1.20±0.48* 2.49±0.47 3.59±0.95 2.56±0.46 3.17±0.55

42 d 3.29±0.62 3.14±0.49 2.05±0.45* 3.29±0.23

48 d 3.79±0.17 3.55±0.55 2.35±0.39* 4.03±0.70

1)

CnmeGV, Cnaphalocrocis medinalis granulovirus; OB, occlusion body; chlorpyrifos, 40% chlorpyrifos at dose of 1 200 mL ha–1; control, treated with water and Tween-80 (0.1%). Data are mean±SE (n=3). * and ** indicate significance at P<0.05 and P<0.01, respectively.

on forest pests, and fewer quantitative studies were available on viral persistence and proliferation where the host feeds on agricultural crops that were periodically harvested, reducing the potential for virus persistence and dispersal (Hajek et al. 2007). In the present study, rice growth experienced a long cycle and suffered from infestation of several generations of C. medinalis. This relative longer period enabled baculoviruses to infect the larvae, persist and disperse among the populations of host. Infection by baculoviruses is generally lethal and therefore has the potential to influence host population densities, particularly if viral transmission increases with host density (Cory and Myers 2003). CnmeGV application caused the infection and death of the occurring larval generation and the release of viruses. The released viruses contaminated the rice leaves, thereby infecting the next field generation of larvae. This resulted in CnmeGV infection not only in larval populations of current generation, but also in the next generation (Fig. 1). Horizontal transmission of baculoviruses is regarded as the main route through which viruses are transmitted among host populations (Elderd 2013). Baculoviruses produce occlusion bodies, in which viral particles were embedded that protect viruses from environmental hazards, including ultraviolet light, allowing the occluded viral particles to survive (Rohrmann 1992). This structure enabled CnmeGV to persist for prolonged periods in the field and transmit to a new host (Zhang et al. 2014). When a susceptible insect ingests food contaminated with OBs, infection from one host to another occurs (Cory and Myers 2003). Baculoviruses are regarded as suitable alternatives to synthetic pesticides because they have high specificity with their hosts, and their application does not directly affect beneficial insects. Thus, baculoviruses are good candidates for biological control and ideal components for integrated pest management system (Szewczyk et al. 2006). Cydia pomonella granulovirus (CpGV) has been commercialized very successfully, it is highly pathogenic and acts rapidly to the larvae of codling moths when applied during the crucial period of egg hatching (Lacey et al. 2008). In this study, we demonstrated the fundamental effects of CnmeGV application to prevent the pest infestation of C. medinalis

in rice fields. CnmeGV spraying during peak egg hatching time was highly virulent, and effectively infected the larvae of C. medinalis. Larval mortality rates increased to 74 and 80% of different concentrations, respectively, at the later stage of current generation. Due to the high mortality rates at larvae stage, the emerging moth density reduced, effectively suppressing the population of rice leaffolder. Moreover, damage of the rice leaves caused by the pest of current and following generations reduced remarkably, suggesting that CnmeGV is a valuable alternative for rice leaffolder management in rice paddies. These results were in agreement with our previous study (Liu et al. 2013). For farmers, killing speed is the most important characteristic and used to determine the efficacy of insecticides in preventing crop damage. From the perspective of conservation control, pest suppression relies on the longterm reproduction or establishment of the pathogens, and the time it takes for the pathogens to replicate and kill their hosts (Landis et al. 2000). In the field applications, larvae infection by CnmeGV required a long time (approximately 12 d) to death. It acted at a significantly slower rate than synthetic pesticides, and resulted in a relatively higher rolled leaf rate than synthetic pesticides. However, at late stage, especially in the next generation, larval density dropped remarkably and no significant difference of leaf rolled rates were found between CnmeGV and synthetic pesticide treatments, which maintained pest infestation at economically acceptable levels. Effective control relies on the primary and secondary cycling of the pathogen to modify the environment by protecting and encouraging natural enemies that are already present within the system (Cory and Franklin 2012). In contrast to synthetic pesticide treatments, the application of CnmeGV was compatible with the arthropod natural enemies of C. medinalis. CnmeGV did not directly affect non-targeted insect species, thereby maintaining the interactions between arthropod insects and their natural enemies and increasing the resilience of cropping systems through their continuous presence (Messelink et al. 2014). This positive interaction resulted in a relatively higher abundance of arthropod natural enemies, persistently

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controlled pests, and maintained the species diversity. It is important to note that the infected larvae, which survived for long periods, might increase the vulnerability of the pests to predation, and the availability of food for their natural enemies, hence conserving their populations (Cloutier and Jean 1998). At the same time, the abundance of the arthropod natural enemies also enhanced the dispersal of entomopathogens (Down et al. 2009). Latency of baculoviruses might be an important component of the system of interactions between them and their host insects, which experience population explosions (Ilinykh and Ulianova 2005). This latent infection can transfer to the acute phase with lethal consequences for insects, thus, becoming a factor in selection and abundance control (Burden et al. 2003). Although CnmeGV was identified in the field in China thirty years ago and was proved to persist in the place for long time, isolation of new strains or prevalence of the virus in the populations of pest was seldom reported. Is there any stage of CnmeGV present in wild populations of host and influenced the density of rice leaffolder must be clarified. Cnaphalocrocis medinalis is a typical migratory pest. If CnmeGV can vertically transmit through latent infection in its hosts, moths infected with latent forms of CnmeGV might carry and diffuse the baculovirus to remote paddies, especially the original site. Such a transmission would significantly influence the population density of rice leaffolder. Under this hypothesis, additional field test and isolation and genetic characterization of existing and novel strain of CnmeGV should be performed.

5. Conclusion Under field condition, CnmeGV infected the larvae of C. medinalis effectively not only in the current generation but also in the successive generation. Due to the sustained infection to pests and the compatibility with arthropod predators, C. medinalis population was suppressed at a low level. These results revealed that CnmeGV is suitable as an agent for rice leaffolder management in rice paddies.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31071740 and 31701792), the Jiangsu Science Foundation of China (BK20181283), and the Jiangsu Agricultural Science and Technology Innovation Fund, China (ZX(17)2002). We are grateful for the experimental sites and technical field assistance provided by Prof. LÜ Zhongxian and Prof. XU Hongxing, Zhejiang Academy of Agricultural Sciences, China.

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