YAN Jun-jie,Shovon Chandra SarkarMENG Rui-xia,Stuart REITZ,GAO Yu-lin

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 College of Agronomy,Inner Mongolia Agricultural University,Hohhot 010019,P.R.China

3 Department of Crop and Soil Sciences,Malheur County Extension,Oregon State University,Ontario,OR 97914,USA

Abstract The entomopathogenic nematode,Steinernema carpocapsae,was evaluated for control of the potato tuber moth,Phthorimaea operculella,under laboratory conditions.We evaluated different concentrations of S.carpocapsae for control of 2nd,3rd,and 4th instar P.operculella.The median lethal concentration (LC50)of S.carpocapsae infective juveniles (IJs)to 2nd,3rd and 4th instar larvae of P.operculella was 200,363,181 IJs mL–1,respectively.With the extension of treatment time,the cumulative mortality increased for 2nd,3rd,and 4th instar larvae and pupae of P.operculella.Fourth instars were the most susceptible for all observation periods.Therefore,our results suggest that S.carpocapsae could be an effective biological control agent for P.operculella.

Keywords:Steinernema carpocapsae, Phthorimaea operculella, patato,integrated biological pest control

1.introduction

The potato tuber moth,Phthorimaeaoperculella(Zeller)(Lepidoptera:Gelechiidae),is a notorious pest of solanaceous crops,especially for potato,tobacco and eggplant,resulting in significant economic losses worldwide (Gao 2018a,b;Xuet al.2019).It is a particularly devastating pest of potato.Larvae mainly infest leaves and petioles in the potato growth phase;while,late in the season,larvae can infest tubers in the ground and theP.operculelladamages tubers seriously during storage (Rondon 2010).Until the last two decades,control ofP.operculellahas relied upon the use of the traditional insecticides (Szendrei 1986;Kroschel 1995);however,insecticide-based control has become difficult due to the rapid development of resistance to insecticides(Do?ramaci and Tingey 2010;Hafez 2011).

Recently,biological control ofP.operculellahas become important in both field production and potato storage(Kepenekciet al.2013).Entomopathogenic nematodes can effectively control a variety of soil-borne pests (Shapiro and Mccoy 2000;Yadav 2012).Steinernemacarpocapsae(Weiser)(Nematoda:Steinernematidae)also is particularly effective against lepidopteran larvae,as well as species that mine within leaves or other protected habitats (Wilsonet al.2012;Kepenekciet al.2013).This study was to investigate the effectiveness ofS.carpocapsaeagainstP.operculellaunder laboratory conditions to provide a basis for development of biological control techniques.

2.Materials and methods

A total of about 500Phthorimaeaoperculellaadults were collected from a potato field in Kunming,Yunnan Province,China (103°79′E,25°5′N(xiāo))in 2014.Phthorimaea operculellawere maintained in culture as described by Rondonet al.(2009).Briefly,the colony was maintained at (27±2)°C,60% RH and photoperiod of 12 h L:12 h D.Larvae were reared in plastic containers.As a food source,a potato tuber with a diameter of about 6 cm was placed in each container,and fine sand was placed in the bottom of the container for pupation.Age is distinguished by the life table ofP.operculellaat 26°C (Jinet al.2005).Pupae were transferred to cylindrical containers for adult eclosion.Cylinders had a diameter of 14 cm and a height of 10 cm.The top of the cylinders were covered with gauze cloth and filter paper (9 cm in diameter)and placed over the gauze as an oviposition substrate.A 5% honey water solution was streaked on the gauze as a food source for adults.

Steinernemacarpocapsaewere provided by Zhejiang Green God Natural Enemy Biotechnology Co.,Ltd.,China and stored in water-soaked sponges held at (10±1)°C.The storage period was not more than 15 days.The nematode was rearedinvivoon late instarTenebriomolitorL.according to the methods of White (1927).Infective juveniles(IJs)ofS.carpocapsaewere collected from cultures at days 12 and 15 of a generation.The storage period of filtered nematodes was used for infestingP.operculella.All laboratory experiments were conducted in Petri dishes(3.5 cm in diameter)held in an incubator at (27±2)°C,60%RH and a photoperiod of 12 h L:12 h D.Ten individuals of the same stage were placed in a Petri dish to comprise an experimental replicate.There were three replicates for each treatment in this experiment.

To screen mortality effects ofS.carpocapsaeonP.operculella,2nd,3rd and 4th instar larvae were treated with one of seven concentrations ofS.carpocapsaeIJs (100,125,250,500,625,1 250 and 2 500 IJs mL–1,respectively).An untreated control of sterile water was also included in the trial.Ten larvae were placed on a 5-mm thick potato tuber disk,and 0.5 mL of the different concentration ofS.carpocapsaesuspension was evenly dropped into the corresponding Petri dish.The potato tuber disk provided nutrition forP.operculella,and also maintained high humidity in the Petri dish.

Insect inactivity after external stimulation was used as an indicator of mortality at 48 h after treatment.At this time,P.operculellawas dissected and the infection ofS.carpocapsaewas observed,and photographs were used for evidence.This information established whether the death ofP.operculellawas due to infestation ofS.carpocapsae.

In the second experiment,P.operculellawas exposed toS.carpocapsaeto evaluate the mortality at 24,48,72 and 96 h after the treatment of 200 IJs mL–1on 2nd,3rd and 4th instars of larvae and pupae.Larvae were held as described above.Pupae were placed in sand at 16% relative humidity.The status of individuals was examined every 24 h after treatment for 4 days.

Mortality data were corrected for control mortality (Abbott 1925).Data were normalized using arcsine square-root transformation before being subjected to analysis of variance(ANOVA).Means were separated by the Tukey’s mean separation test at α=0.05 to determine significant differences among treatments.The median lethal concentrations (LC50)were determined by Probit regression.All analyses were conducted using the SPSS 19.0 Software.

3.results

Steinernemacarpocapsaewas lethal to the different stages ofP.operculellalarvae.There were significant differences in LC50among the different stages ofP.operculella(F=65.01;df=2,6;P<0.0001).Fourth and 2nd instars were the most sensitive (LC50=181 IJs mL–1;LC50=200 IJs mL–1,respectively);3rd instar larvae were the least sensitive(LC50=363 IJs mL–1)(Table 1).

There were significant differences in mortality over time (F=49.312;df=3;P=0.001)and between life stages(F=19.145;df=3;P=0.001)level.Fig.1 shows that the mortality ofP.operculellaincreased with the time after treatment.For example,mortality of 4th instarP.operculellaat 24,48,72 and 96 h was 6.67,44.44,92.58 and 98.89%,respectively.Third and 4th instars had similar mortality rates over the 4 days of the experiment.Second instars were less susceptible toS.carpocapsaethan 3rd and 4th instars.Pupae were the least susceptible toS.carpocapsae(Fig.1).Dissection of putatively infectedP.operculellarevealed thatS.carpocapsaecan infect and reproduceinvivoinP.operculella(Fig.2).

4.Discussion

In Southwest China,P.operculellaroutinely infests 20 to 30%of tubers in the field and up to 100% infestation in storage (Duet al.2006).The study which combine climatic phenology model forP.operculellawith global climate change clearly indicates that the invasiveness of this pest will continue to the north from south as climatic temperatures continue torise (Kroschelet al.2013).The increase ofP.operculellawill severely threaten the development of the potato industry.Numerous studies have been conducted on the efficacy of entomopathogenic nematodes againstP.operculellalarvae and pupae.Most of these studies have indicated that the larvae are susceptible for nematode infection,whereas pupae not (Ivanovaet al.1994;Hassanikakhkiet al.2013;Kamaliet al.2013;Kepenekci˙et al.2013;Karyet al.2018).Our results indicate thatS.carpocapsaeis highly virulent toP.operculellalarvae and it significantly reduces the survival ofP.operculellalarvae.Steinernema carpocapsaecauses higher larval mortality and 4th instar is the most susceptible stage ofP.operculella.Mortality ofP.operculellaincreased with time following treatment.Our results also demonstrated thatS.carpocapsaecasued higher mortality than observed in laboratory trials for other heterorhabditid nematodes(Yoshida 2010;Vashisthet al.2018).

Table 1 Median lethal concentrations (LC50)of Steinernema carpocapsae against different stages of Phthorimaea operculella larvae

Fig.1 Cumulative mortality for different instars of Phthorimaea operculella (2nd,3rd,4th instars and pupae)following exposure to Steinernema carpocapsae (200 infective juveniles mL–1).Mortality (±SE)of P.operculella larvae and pupae were treated by S.carpocapsae in the laboratory.Means with different letters are significantly different (P<0.05).

Fig.2 Steinernema carpocapsae invading the larvae of Phthorimaea operculella and multiplying within the hosts’ bodies after treatment.The red arrows refer to Steinernema carpocapsae which infected into P.operculella larvae,while the blue arrow refers to S.carpocapsae that propagated in P. operculella larvae.

The results of this research are especially encouraging as the laboratory bioassay has indicated high infectivity against the larval stages ofP.operculella.Applications ofS.carpocapsaecould be particularly promising in targeting the 4th instars,which is the stage at which larvae prepare to pupate at or near the soil surface in the field.Therefore,further research needs to be conducted in field conditions to support our laboratory results regarding the virulence ofS.carpocapsae.In addition,given thatP.operculellacauses major damages in a suitable climates.Steinernema carpocapsaerequires a minimum of 6%soil moisture,but survival rate of these nematodes drops significantly at higher soil moistures (Koppenh?feret al.1995;Yadav 2012).Therefore,more attention should be paid to the limitations ofS.carpocapsaein pluvial regions,which are regions whereP.operculellais a greater problem.

Steinernema carpocapsaeshows strong virulence toP.operculellalarvae,and can be reproducedinvivoinP.operculellain the laboratory conditions.After successful infection,it may be expected to form reproducing,persistant populations to provide long-term management ofP.operculellain the field.

5.Conclusion

Our study indicates thatS.carpocapsaemay be recommended for effective biological control ofP.operculellaand further research needs to be done in open field experiments to support our laboratory studies regarding the efficacy ofS.carpocapsae.

Acknowledgements

This work was supported in part by the National Key Research and Development Program of China (2018YFD0200802).