Abstract:

【Objective】Cnaphalocrocis medinalis granulovirus (CnmeGV) is a specific pathogenic microorganism of Cnaphalocrocis medinalis. Persistent infection of baculovirus plays an important role in pest population control. The objective of this study is to construct the detection method of persistent infection with CnmeGV, analyze the control effect of viral persistent infection on pest population, and to provide a theoretical basis for the application of viral pesticides.【Method】The largely diverged regions were selected to design CnmeGV nested PCR primers based on multiple comparisons of granulin gene sequences of granulovirus. The sensitivity and reliability of probes were further evaluated. The maize leaves were used to feed C. medinalis in the laboratory. The 4th instar larvae were singled out, and then fed with maize leaves soaked by CnmeGV with a sublethal concentration of 10⁶ OB/mL. The infected C. medinalis were reared to adult stage, and the emergence rate was counted. After treating the insect body surface with 10% formaldehyde, the carrying CnmeGV rates of larvae, pupae, pupal molts and adults in persistent infection population of C. medinalis were analyzed using these probes. The prevalence rate of larvae and carrying rate of soil in the next year after using CnmeGV were also detected.【Result】The probes of CnmeGV nested PCR were constructed including the outer primer Cm-gran1 and the inner primer Cm-gran2. The sensitivity of nested PCR was 0.85 fg·μL ^-1 genomic DNA, which was 1 000 times higher than that of the conventional PCR. The probes were highly reliable, and no target fragment was detected in the food sources of C. medinalis (rice and maize), other polyhedrosis and granulosis viruses. The target fragments of CnmeGV were not detected in larvae after treated with 10% formaldehyde for 10, 30 min and 16 h, respectively. However, the pupa of C. medinalis could not emerge after 16 h of treatment. The detection rate of CnmeGV was 100% in the larvae of infected population treated with sublethal concentration of CnmeGV for 96 h. After pupation and emergence, the detection rates of pupae, pupal molts and adults were 87.5%, 83.3% and 16.7%, respectively. The detection rate of CnmeGV had no significant change from larva to pupa stage (χ ²=3.2, P=0.234) by Chi-square test. However, the detection rate of CnmeGV decreased significantly (χ ²=32.356, P=0) from pupa to adult stage, indicating that most of the viruses were excreted from the body with pupal molt during metamorphosis. The emergence rate of adult in infected population (30.8%) was significantly lower than that of the control (93.4%). Field tests showed that the prevalence rate of C. medinalis larvae in the field at one year following CnmeGV treatment was 4%, and the rate of soil carrying CnmeGV was 58%, suggesting that CnmeGV could survive in the soil and continue to infect C. medinalis through horizontal transmission.【Conclusion】The sensitivity of constructed nested PCR probes is high and can be used for the detection of CnmeGV persistent infection. Persistent infection of CnmeGV can effectively control the population development of C. medinalis, and adult metamorphosis plays an important role in removing pathogens.

Key words: Cnaphalocrocis medinalis, granulovirus, persistent infection, latent infection, molecular marker, horizontal transmission, insect metamorphosis