Vuill. and Steinernema Feltiae (Filipjev) on the Pronymphs and Eonymphs of Cephalcia Tannourinensis (Chevin) Under Laboratory Conditions

Research Paper

Efficacy of local strains of entomopathogenic Beauveria bassiana (Bals.) Vuill. and Steinernema feltiae (Filipjev) on the pronymphs and eonymphs of Cephalcia tannourinensis (Chevin) under laboratory conditions

Accepted 15th January, 2019

ABSTRACT

The Lebanese web spinning cedar sawfly, Cephalcia tannourinensis (Chevin), is the main cedars defoliator in Lebanon. The study is to determine the effectiveness, competition and the host finding ability of two indigenous entomopathogens; a fungi Beauveria bassiana (Balsamo) and a nematode Steinernema feltiae (Filipjev) under laboratory conditions. The study included also the use of a commercial entomopathogenic nematode Heterorhabditis bacteriophora (Poinar). The study targeted the two diapauses stages of the cedar sawfly, the pronymphs, and the eonymphs. One dose of the entomopathogenic nematodes and two doses of the entomopathogenic fungi were applied as well as, mixtures of the two. Local strain of B. bassiana caused mortalities of 60 and 53% on the pronymphes when applied at 50 and 500 spores/larvae, respectively. A synergistic effect was proven when the treatment consisted of two mixtures of the entomopathogens, B. bassina and S. feltiae / H. bacteriophora, and caused mortalities of 100% on the pronymphs and 86.6 to 100% on the eonymphs. The host finding ability was higher for the C. Al Khoury1, M. Rehayem2, C. Abou Jaoudé1, entomopathogenic nematode than for the entomopathogenic fungus. The use of a E. Noujeim2 and N. Nemer1* combination of entomopathogens proved to be more effective when only one is

1Department of Agricultural Sciences, Faculty used. The local strain of B. bassiana when used at the dose of 500 spores/larvae of Agricultural and Food Sciences, Holy Spirit was effective. University of Kaslik, P.O.Box 446, Jounieh, Lebanon. Key words: Cephalcia tannourinensis, biological control, Beauveria bassiana, 2National Council for Scientific Research - CNRS, P.O.Box 11-8281, Ryad El Solh 1107 Steinernema feltiae. 2260, 59, Zahia Selman street, Beirut, Lebanon. Abbreviations: LIB132: Steinernema feltiae; Hb.Com: Heterorhabditis bacteriophora; USEK: Holy Spirit University of Kaslik; CNRS: National Council for *Corresponding author. E-mail: [email protected]. Tel: 961 9 600893; Scientific Research; PDA: Potato Dextrose Agar; SPSS: Statistical Package for the Fax: 961 9 600871. Social Sciences; ANOVA: Analysis of variance.

INTRODUCTION

The cedar sawfly, Cephalcia tannourinensis has been pronymphs are the larvae that are ready to become nymph causing severe damages to the cedars of Lebanon since and emerge as adults the following year. The eonymphs are 1992 (Nemer et al., 2005). One of the particularities of this the larvae that are still in diapause and may emerge in the insect species is that it evolved physiologically and timing coming two to seven years; this feature is present in several mechanisms such as diapauses in order to emerge at an Cephalcia species whereby eonymphs can persist in the soil appropriate time to utilize resources even if they have to up to 5 years (Nemer et al., 2014; Gruppe, 1996). face adverse environmental conditions (Danks, 1987). C. Following its outbreak in 1998, this pest was treated with tannourinensis has two diapausing larval stages, the an insect growth regulator, diflubenzuron, which is no Academia Journal of Agricultural Research; Khoury et al. 055

longer homologated in forest management (Nemer et al., hemocytometer method. Spores were harvested from Petri 2005). The search for alternatives to chemical pesticides dishes cultures of the fungus grown on potato dextrose agar has been under investigation since 2007 (Nemer et al., by flooding the culture with sterile water and dislodging 2007; Abdo et al., 2008; Noujeim et al., 2010). spores from the hyphen with the aid of a sterile loop. The In Lebanon, the entomopathogenic fungus, Beauveria spore solution was then filtered through four consecutive bassiana, was found in the forest Tannourine Hadath El sterile layers of cheesecloth to remove any hyphen Jebbeh (North Lebanon) growing on C. tannourinensis fragments present. The number of spores can be counted diapausing larvae in the soil. The results of a study using a hemocytometer. conducted by Abdo et al. (2008) showed that B. bassiana is Two nematode strains were tested, the indigenous strain a natural entomopathogenic fungus parasitizing the larvae Steinernema feltiae (LIB132) and a commercial strain of C. tannourinensis and has been demonstrated to cause Heterorhabditis bacteriophora (Hb.Com). high mortalities of eggs and larvae of the sawfly under laboratory conditions. However, this fungus has not been applied to the soil diapausing stages of the cedar sawfly. Experimental method The entomopathogenic nematode, Heterorhabditis was also found in Tannourine Hadath El Jebbeh cedar forest Experimental tests were carried out in November, 2017 in (Noujeim et al., 2010) and was used against the diapausing the Laboratory of Entomology at the Faculty of Agricultural stages of C. tannourinensis in vitro (Noujeim, 2010; and Food Sciences – USEK. The soil was sampled from the Rehayem et al., 2018). Species of entomopathogenic Tannourine Cedar Nature Reserve and then transferred nematodes Steinernema feltiae, Steinernema kraussei into jars and sterilized in an autoclave at 120°C for 20 min (Steiner) were also proposed as biological agents to fight to kill all possible micro-organisms that may interfere with against Cephalcia arvensis (Panzer) (Battisti and Masutti, the effect of the tested entomopathogens. The soil was 1994; Führer and Fischer, 1991). poured in sterilized 100 ml plastic contained and closed Two hypotheses were made: the first was to check with a lid. The eonymphs and pronymphs were then whether the annual diapauses stage (pronymphs) of C. transferred to the sterilized soil. tannourinensis behaves differently from the multi-year The treatments consisted of two concentrations of the diapausing stages (eonymphs). The second assumption is local strain of the entomopathogen B. bassiana (50 that entomopathogenic fungus and nematodes have spores/larvae and 500 spores/larvae); one concentration different search behavior from each other implying a of the commercial entomopathogenic nematode H. synergy between the two agents that will increase their bacteriophora (HB.com) supplied by Koppert; one efficiency. concentration of the local strain of the entomopathogenic Therefore, the first objective of this study was to nematode S. feltiae (LIB132) and three mixtures of both determine the effectiveness of these two entomopathogenic entomopathogens; in addition to a positive control fungi and nematode on the two stages of diapauses of the consisting of Tween 80 and one control without any cedar sawfly under laboratory conditions. The second addition. Table 1 represents all the dosages and treatments objective was to determine the search behavior for both administered to the two diapausing stages of the C. entomopathogenic fungus and nematode. tannourinensis. The larvae were separated into pronymphs and eonymphs put separately in a sterile beaker prior to the MATERIALS AND METHODS application of different treatments (Table 1). Five larvae of each stage (pronymphs and eonymphs) were placed Animal material separately within one container and each treatment replicated thrice. The eonymphs and pronymphs of the cedar sawfly C. A surfactant Tween 80 was added to the spores’ solution tannourinensis were sampled from the cedar forest of of B. bassiana to increase its adhesion effect. Using an Tannourine in October, 2017. The sampled larvae were Eppendorf micropipette, the different treatments at the transferred to the laboratory of Entomology at the Holy indicated doses were applied topically on the pronymphs Spirit University of Kaslik (USEK). Thereafter, they were and eonymphs larvae of C. tannourinensis. The total number separated into eonymphs and pronymphs based on the of pronymphs and eonymphs larvae for each treatment was compound eyes feature (Nemer et al., 2014). 15, while the total number of pronymphs and eonymphs The entomophagous fungus, B. bassiana was isolated larvae used in this study was 300. from dead larvae of C. tannourinensis which was cultured Three days after the application of the different on potato dextrose agar (PDA) medium. Conidia suspension treatments, the larvae of the following treatments (B. of B. bassiana was prepared in sterile water and filtered to bassiana 50 and 500 spores/larvae, Tween, and control) remove any mycelia fragments. The concentration of spores were removed from containers and transferred to PDA in the suspension was determined by Neubauer culture media on petri dishes. Larvae that are already dead Academia Journal of Agricultural Research; Khoury et al. 056

Table 1: Treatments and doses of entomopathogens.

Treatments Doses Beauveria bassiana (local strain) 50 spores/larvae + 1% Tween Beauveria bassiana (local strain) 500 spores/larvae + 1% Tween Heterorhabditis bacteriophora (HB.Com) 250 juvenile nematodes/larvae Steinernema feltiae (LIB132) 250 juvenile nematodes/larvae HB.Com + Beauveria bassiana 50 spores/larvae + 1% Tween + 250 juvenile nematodes/larvae HB.Com + Beauveria bassiana 500 spores/larvae + 1% Tween + 250 juvenile nematodes/larvae LIB132 + Beauveria bassiana 50 spores/larvae + 1% Tween + 250 juvenile nematodes/larvae LIB132 + Beauveria bassiana 500 spores/larvae + 1% Tween + 250 juvenile nematodes/larvae Tween 1% Control -

were recorded, while mortalities and mycelial growth were P>0.05). examined every week for three weeks on the culture media. One week after treatment, the effects of different The dead larvae treated with nematodes or with a treatments on pronymphs and eonymphs showed a mixture of the two entomopathogenic agents were significant difference on both factors: diapauses stage and transferred in a nematode Dutky white trap (Dutky et al., treatment type. Table 2 presents mortality rates. Eonymphs 1964), a harvesting device to determine the cause of death. of C. tannourinensis were more susceptible to the This device consists of a large petri dish (diameter of 9 cm), treatments than the pronymphs one week after treatment in the center of which is placed a reversed smaller petri with B. bassiana, commercial nematode HbCom, indigenous dish (diameter of 5 cm) to create a relief. The smaller petri nematode Lib132, and of nematodes-Beauveria mixes. dish was covered with a filter paper that overflows into the One-week post application, treatments with B. bassiana big petri dish. Ringer's solution was introduced (liquid caused mortality of 6.67 and 20%, respectively for the similar to that of the internal medium of an organism, pH = doses of 500 and 50 spores/larvae for pronymphs and were 7.2) which by capillarity wets the entire filter paper. The not significantly different from the results of the control dead larvae were then placed on the wet filter paper (0%) and the Tween (13.33%) (Table 2). Treatments with covering the small petri dish. Third stage juveniles the mix of commercial nematode (HbCom) with the nematodes can then escape from their host and migrate entomopathogen fungi B. bassiana at a dose of 500 into the bigger petri dish through the filter paper soaked spores/larvae was more efficient and caused mortality rate with the Ringer’s solution after a week. Two readings of of 93.33% (Table 2). Similarly, all treatments with a mix of mortality were recorded within an interval of one week. B. bassiana and nematodes caused mortalities that were The subject larvae that produced juvenile nematodes in the significantly different than the control (Table 2). Ringer’s solution died because of the nematode larvae Commercial nematode Hb.Com applied alone caused a while the one with external sporulation died because of the mortality rate of 80% and was more efficient thant the fungus B. bassiana. indigenous nematode LIB132. All mortalities were recorded, and the experimental Regarding eonymphs, and one week following design analyzed using two-way ANOVA (Analysis of application, all treatments caused mortalities that were variance) for treatments and diapausing stage using the significantly different between the control and the Tween statistical package SPSS (Statistical Package for the Social (F=7.14, df=9, P<.0.05) (Table 2). Mortality rates caused by Sciences). B. bassiana were higher than those registered in pronymphs. The highest mortality rates in eonymphs registered were due to the commercial nematode HbCom RESULTS treatment at a dose of 250 J/larvae 93.33%, (Table 2) and the mix HbCom and B. bassiana at 500 spores/larvae. These Efficacy of treatments two treatments were also the most efficient against the C. tannourinensis pronymphs (Table 2). The diapausing stage of C. tannourinensis had a significant Two weeks after application, results obtained were also effect on the mortalities observed, 7 days after treatment affected by the diapausing stage and the type of treatment (F=11,25, df=1: 19, P<0.05) as well as, 14 days after applied against pronymphs and eonymphs (F=6.76, df=1: treatment (F=6.76, df=1: 19, P<0.05). This difference is no 19, P<0.05; F=28.95, df=1: 19, P<0.05). Treatments longer found 21 days after treatment (F=0.8, df=1; 19. consisting of a mix of the two types of entomopathogens Academia Journal of Agricultural Research; Khoury et al. 057

Table 2: Effect of the different treatments on the mortality of eonymphs and pronymphs of Cephalcia tannourinensis.

Three weeks One week after application Two weeks after application after application

Treatments Cumulative of Pronymphs Eonymphs Pronymphs Eonymphs pronymphs and eonymphs Mortality (%) ± S.E 20.00 ± 60.00 ± 60.00 ± 28.3 Beauveria† 50 spores 80.00 ± 16.3b* 70.00 ± 22.3b* 16.3a* 16.3ab* a* Beauveria 500 spores 6.67 ± 9.4a 53.33 ± 33.9ab 40.00ab 86.67 ± 18.8b 76.67 ± 4.7bc HbCom‡ 80.00 ± 16.3c 93.33 +9.4b 86.67 ± 9.4cd 93.33 ± 9.4b 90.00 ± 9.4bcd Lib132§ 26.67 ± 9.4ab 66.67 ± 9.4 b 53.33 ± 9.4bc 80.00b 93.33 ± 4.7cd HbCom + Beauveria 50 73.33 ± 9.4c 80.00b 93.33 ± 9.4cd 80.00b 90.00bcd spores HbCom + Beauveria 500 93.33 ± 9.4c 93.33 ± 9.4b 93.33 ± 9.4cd 100.00b 100.00d spores Lib132 + Beauveria 50 66.67 ± 9.4bc 86.67 ± 18.8b 86.67 ± 9.4cd 86.67 ± 18.8b 93.33 ± 4.7cd spores Lib132 + Beauveria 500 80.00 ± 16.3c 66.67 ± 24.9b 100.00d 100.00b 100.00d spores Tween 80 13.33 ± 9.4a 6.67 ± 9.4a 26.67 ± 9.4ab 20.00 ± 9.4a 23.33 ± 4.7a Control 0.00a 6.67 ± 9.4a 6.67 ± 9.4a 13.33 ± 9.4a 10.0 ± 9.4a

*Means followed by the same letter within a column are not significantly different at P<0.05. † Beauveria: Beauveria bassiana, local strain; ‡ HbCom: Heterorhabditis bacteriophora, Commercial; Lib132: local strain of Steinernema feltiae.

(Beauveria and nematode) are the first to achieve a and entomopathogenic nematodes were efficient and mortality rate of 100%. Eonymphs are more susceptible to caused mortalities of 80 and 86.67% that were not treatments by the entomopathogen B. bassiana than the significantly different from the other treatments except for pronymphs. The mix of indigenous nematode Lib132 and B. control and Tween. bassiana (500 spores/larvae) was the most efficient and Three weeks post application, the diapausing stage had caused a mortality rate of 100% in the pronymphs. This no more effect on mortality rates (F=0.8, df=1: 19, P>0.05), treatment along with the other mixes of the two however, the treatment had a significant effect (F=46.47, entomopathogens caused a mortality rate that is df=1: 19, P<0.05). Treatments with the mixtures of statistically comparable for both doses of B. bassiana (50 entomopathogens were the most efficient and caused a and 500 spores/larvae) (Table 2). B. bassiana treatment at mortality rate of 100% in the pronymphs and 80 to 100% a dose of 500 spores/larvae caused a mortality rate of 40% in eonymphs, respectively (Table 2). which is comparable to the Tween treatment and to the Since the treatment type is the only factor to have control test; however, the treatment of B. bassiana at a dose affected mortality rates independently of the diapauses of 50 spores/larvae caused a significantly higher mortality stage, the results of both stages were pooled together to rate of 60% (Table 2). calculate the average mortality rate induced by each Two weeks after treatment, the eonymphs mortalities treatment (Table 2). The mixes of two entomopathogens caused by entomopathogens treatments were comparable resulted in the highest mortality rates, especially with the and significantly different from the control and of the doses of 500 spores/larvae of B. bassiana with 250 J/larvae Tween (F=16.83, df=9, P<0,05) (Table 2). Treatments of either the commercial nematode (HbCom) or the containing the commercial nematode HbCom mixed with B. indigenous (Lib132) (F=46.47, df 9: 19, P<0.05). Both doses bassiana (500 spores/larvae) as well as, the mix Lib132 of B. bassiana caused a mortality rate of 70 and 76.67% for with B. bassiana (500 spores/larvae) were the most the doses of 50 and 500 spores/larvae, which were efficient with a mortality rate of 100%. The two other significantly different from those of the control and Tween treatments with mixes of B. bassiana (50 spores/larvae) treatments. Academia Journal of Agricultural Research; Khoury et al. 058

Identification of the causing agent of mortalities within is in favor of the nematode in the experiments. pronymphs and eonymphs when using mixed Direct exploitation of living organisms to control pests treatments of both entomopathogens requires an understanding of the mechanisms of interaction between the entomopathogenic agent and the target pest. Dutky tests used to determine the reason of mortality Indeed, the pathogenicity of the inoculum and the revealed that mortalities in treatments containing both specificity of the host are two important parameters in the entomopathogens were mainly due to the presence of choice of the fungal isolate. B. bassiana proves to be an nematodes (Tables 3 and 4). In pronymphs, nematodes interesting control agent since it has the advantage, were the main cause of mortality for all 4 treatments compared to other pathogenic micro-organisms, of comprising a mix of both entomopathogens (Table 3). infecting the host without being ingested. This may make Mortalities due to nematodes were also higher in the different stages of host development susceptible to this eonymphs; B. bassiana however contributed with biopesticide. This feature also ensures that this fungus can percentages varying from 13.33 to 46.67% of the total be effective against stinging-sucking pests that are eonymphs mortality (Table 4). Larvae tested by Dutky relatively free from infection by spores deposited on plant revealed that one cause for mortality is when larvae are foliage (Wraight and Roberts, 1987). infested with entomopathogenic nematodes and no The fungus species of the genus Beauveria have development of B. bassiana fungus was observed and vice considerable agronomic interest in the biological control of versa. crop pests and are therefore being extensively studied. In addition, the persistence of conidia in soil may provide long-term control by causing a fungal infection in DISCUSSION subsequent generations of the host (Gaugler and Lashomb, 1989). In terms of biosecurity, several studies have shown The results observed, one-week post application proved that this fungus is not dangerous to vertebrates (Faria and that the entomopathogen nematodes have a faster effect Wraight, 2001). than B. bassiana on C. tannourinensis pronymphs and The mortalities with B. bassiana doses of 50 and 500 eonymphs and that the two-combined work in synergy spores/larvae were less virulent on the diapausing stages since mortality rates of pronymphs and eonymphs of the cedar sawfly as compared with the mortalities increased when treated with mixed doses of the two observed on the aerial stages of C. tannourinensis obtained entomopathogens (Table 2). Nematodes have a higher by Abdo et al. (2008). This could be explained by the fact efficacy mechanism in the search for the host than the fungi that in the experiments conducted by Abdo et al. (2008), since they cause high mortality rates from the first week the larval stages that were considered were the first, after application. These results are in conformity with those second and third larval developmental stages which obtained by Vanninen and Hokanen (1997) who obtained develop on the needles of the cedars and are not in a higher virulence levels in nematodes than in B. bassiana diapausing stage in the soil. In the bioassays conducted in when applications were conducted directly in the soil this experiment, a reproduction of the soil condition of the against the Argyresthia conjugella pest (Zeller). These forest was made under laboratory conditions and showed results concur with those obtained by Shapiro-Ilan et al. that the efficacy of B. bassiana is reduced when applied to (2004) using treatments consisting of the entomopathogen diapausing stages. fungi B. bassiana alongside the nematode Steinernema In this study, two nematodes species were used: the carpocapsae (Weiser) against the weevil Curculio cayae commercial nematode H. bacteriophora (Hb Com) and the (Horn). indigenous strain extracted from the soil of the Tannourine The fact that eonymphs of C. tannourinensis were more forest S. feltiae. These two nematodes showed differences, susceptible to B. bassiana than pronymphs could be especially in infections. The commercial strain H. attributed to the behavior of the pronymphs that are much bacteriophora (Hb Com) caused higher mortalities in more active than eonymphs resulting in them losing a treatment mixtures of the two entomopathogens than the fraction of the spores applied when they came into contact native strain S. feltiae. This could be due to the ambush with the soil. strategy called "cruiser" to hunt their prey in depth which is Therefore, we can conclude that there is a stronger effect a feature of Heterhabditis compared to Steinernema of mixed treatments when the dose of B. bassiana is 500 (Gaugler et al., 1997). The synergistic effects resulting from spores/larvae. This effect fades when the dose of B. the combination of entomopathogenic nematodes with bassiana is 50 spores/larvae, and mortality rates of the other entomopathogenic organisms have been documented mixes become equal to those of the nematodes when in several publications (Shapiro-Ilan et al., 2004). However, applied alone. other publications have reported poor synergistic or even The results obtained by Dutky test imply a competition antagonistic effects between entomopathogenic nematodes between the two entomopathogens and this competition is and other entomopathogens (Brinkman and Gardner, in favor of the nematode. Thus, the host finding mechanism 2000). Academia Journal of Agricultural Research; Khoury et al. 059

Table 3: Mortality rates of pronymphs of Cephalcia tannourinensis according to the causing agent for different treatments.

Total Mortality due to Mortality due to Treatments mortality B. bassiana (%) nematodes (%) (%) HbCom × Beauveria 50 100 0 100 spores HbCom × Beauveria 500 100 13.33 86.67 spores Lib132 × Beauveria 50 100 20.00 80.00 spores Lib123 × Beauveria 500 100 20.00 80.00 spores

Table 4: Mortality rates of eonymphs of Cephalcia tannourinensis according to the causing agent for different treatments.

Total mortality Mortality due to Mortality due to Treatments (%) B. bassiana (%) nematodes (%) HbCom × Beauveria 50 spores 100 13.33 86.67 HbCom × Beauveria 500 spores 100 26.67 73.33 Lib132 × Beauveria 50 spores 100 46.67 53.33 Lib123 × Beauveria 500 spores 100 20.00 80.00

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