Efficacy of Entomopathogenic Fungi with Insecticides Mixtures against Oxycarenus hyalinipennis (Costa) (Lygaeidae: Hemiptera)

Cotton, Gossypium hirsutum L. is a critical fiber crop and exportable produce of Pakistan which contributes 7.0 % to the national economy and 1.7% of the Gross Domestic Product (GDP) of Pakistan (Farooq, 2014). There are numerous factors which contribute for the low cotton production, while insect pests including chewing and sucking are the major factor for lowering the yield of cotton (Shah, 2014; Akram et al., 2013; Sammaiah et al., 2012; Patil and Rajankanth 2005). Yunus and Yousuf (1979) stated that nearly 93 different insects and mites attack on cotton crop that damage the cotton crop in different ways either by sucking the cell sap or eating different parts of plant, causing 20-40% yield loss (Aslam et al., 2004).

The use of insecticides is the common strategy adapted by the farmers for control of Oxycarenus hyalinipennis. About 90% farmers use insecticides against insect pests to protect their crops (Prayogo et al., 2005). However, the unnecessary use of insecticides create problem including resistance (Wang et al., 2011). Owing to these reasons, alternate methods of insect pest’s management including biological control using insect pathogenic organisms have several advantages including low costs, safe for beneficial insects, more efficient and eco-friendly than the conventional insecticides (Carruthers and Hural, 1990; Lacey et al., 2001; Freed et al., 2012). Fungal biocontrol agents have different mode of action than other micro-organisms by invading their hosts directly through cuticle rather than ingestion. This is the reason that insect pathogenic fungi can infect eggs (Ujian and Shahzad, 2007; Anand and Tiwary, 2009) and pupae of the insects (Nguyen et al., 2007; Anand et al., 2008).

Another strategy to increase the efficacy of entomopathogenic fungi is the combined application with insecticides. The studies report that the insecticides application increased the efficacy of fungus making these bicontrol agents more effective for pest management (Quintela and McCoy, 1998; Dayakar et al., 2000; Hiromori and Nishigaki, 2001; Serebrov et al., 2005; Purwar and Sachan, 2006; Rachapa et al., 2007). Combined use of insecticides and entomogenous fungi can increase the efficacy by reducing the quantity of insecticides, minimizing the environmental hazards and resistance in pests (Moino and Alves, 1998; Quintela and McCoy, 1998). Keeping in view the importance of emerging economic insect pests in cotton, the study was planned to evaluate the efficacy of mixtures of entomopathogenic fungi with insecticides against O. hyalinipennis.

The nymphs and adults of dusky cotton bug O. hyalinipennis were collected from the cotton fields. These insects were reared in plastic jars on cotton leaves in the laboratory. The jars openings were covered with muslin cloth. Cotton seeds were also placed in the jars as egg laying medium. Rearing of insects was carried out at temperature 28±1°C, relative humidity 60-70% and 14:10 light: dark photoperiod.

Isolates of Metarhizium anisopliae (Ma-11.1, Ma-2.4, isolated from soil), Isaria fumosorosea (If-03, If-02, isolated from soil and Rove beetle) and Beauveria bassiana (Bb-01, Bb-10, isolated from soil) were used from the laboratory maintained culture. The required concentrations i.e., 1×106- 3×108 conidia/ml of each isolate were made by serial dilution method.

Insecticides, bifenthrin, lambda cyhalothrin, nitenpyram, imidacloprid, triazophos, and pyreproxifin were used to check the efficacy against O. hyalinipennis. Required concentrations of each insecticide i.e., 0.005, 0.0025, 0.00125, 0.000625 and 0.00031ppm were prepared by serial dilution method.

The desired concentrations (LC40, LC30, LC20 and LC10) of fungi and insecticides were calculated by conducting preliminary experiments (Table I). Binary mixture of insecticides and entomopathogenic fungi was made by mixing the LC10 fungi + LC10 insecticides, LC20 fungi + LC20 insecticides, LC30 fungi + LC30 insecticides and LC40 fungi + LC40 insecticides.

The experiment was conducted under complete randomized design with four replications having ten insects per replication. Immersion method was used in which 5th instar nymphs of O. hyalinipennis were individually immersed in the combined concentrations of entomopathogenic fungi and insecticides for 8-10 seconds. To soak up the excessive moisture the insects were placed on tissue paper. While, in the control treatment, insects were treated with Tween 80 (0.05%) solution only. These treated insects were then transferred into petri dishes provided with fresh cotton leaves. The mortality data was recorded for consecutive 7 days. After seven days the remaining alive insects were reared again and were kept under examination to assess the effect of binary mixture on the survival and biological parameters of O. hyalinipennis.

All the values (LC10, LC20, LC30, and LC40) of insecticides and entomopathogenic fungi were calculated by Probit analysis by using POLO-PC software (LeOra Software, 2003). The means were compared by LSD test at 0.05 probability levels and analyzed by analytical software (Statistix version 8.1).

Mortality of 5th instar nymphs of O. hyalinipennis after the application of mixtures of entomopathogenic fungi and insecticides are presented in Table II. An increasing trend of mortality was found towards higher concentration of insecticides and fungi combinations in a dose dependent manner. In case of mixtures of insect pathogenic fungi and insecticides, the highest percent mortality (±SE) (90.0± 4.0) was recorded in case of combined use of higher doses of isolate Bb-01 (LC40) and bifenthrin (LC40,) and Bb-10 (LC40) with bifenthrin and triazophos followed by the LC40 of Bb-01 and LC40 of nitenpyram and LC40 of isolate Ma-11.1 (LC40) and LC40 of triazophos.

The results regarding entomopathogenic fungi (LC30) and insecticides (LC30) mixture showed highest percent mortality (±SE) (82.5 ± 4.7) in Bb-01(LC30) and bifenthrin (LC30) while in case of entomopathogenic fungi (LC20) and insecticides (LC20) mixture, highest percent mortality (75.0 ±6.4) was caused by Bb-01 (LC20) and bifenthrin (LC20). Moreover, in case of LC10 highest percent mortality (75.5 ± 4.7) was observed for If-03 (LC10) and bifenthrin (LC10).

Table II.- Percent nymphal mortality of O. hyalinipennis after treatment of binary mixture of entomopathogenic fungi and insecticides.

Table III.- Nymphal duration of O. hyalinipennis after treatment of binary mixture of entomopathogenic fungi and insecticides.

Table IV.- Percent adult emergence of O. hyalinipennis after treatment of binary mixture of entomopathogenic fungi and insecticides.

Table V.- Male longevity of O. hyalinipennis after treatment of binary mixture of entomopathogenic fungi and insecticides.

The binary treatment of fungi and insecticides significantly prolonged nymphal duration in all treatments. The maximum prolongation of nymphal duration (days) (15.1 ± 0.3) was observed in treatment with Ma-2.4 and bifenthrin (LC40). In case of LC30, nymphal duration (13.1 ± 0.8) days was recorded in treatment with LC30 of Ma-2.4 and nitenpyram. On the other hand, the nymphal duration of 11.8±0.6 days was observed in the treatment of combined mixture of Ma-11.1 (LC20) and nitenpyram (LC20), while 9.9±0.4 days were recorded by the treatment of combined mixture of LC10 of Ma-2.4 and lambda cyhalothrin (Table III).

High doses of fungi and insecticides mixture decreased the adult percent emergence to a greater extent, while significant decrease was also recorded in other doses. The highest percent emergence i.e., 30.0±5.8 and 30.0±4.9 after application of LC40 of Ma-2.4 + imidacloprid and LC40 of If-03 + imidacloprid was recorded, respectively. While the lowest percent emergence i.e., 10.0±2.5 and 10.0±1.8 with the mixture of LC40 of Bb-01 and bifenthrin and Bb-10 and bifenthrin, respectively. In case of LC30 lowest adult emergence (17.5±5.6) was observed in the mixture of LC30 of Bb-01 and bifenthrin. While for LC20 and LC10 lowest adult emergence i.e., 25.0±2.7 and 35.0±3.1 were recorded, respectively in combination with Bb-01 and bifenthrin (Table IV).

The combinations of fungi and insecticides greatly shortened the male longevity. At LC40 and LC20 of Ma-2.4 and imidacloprid reduced the male longevity to 2.8±0.3 and 5.9±0.7 days, respectively. The minimal male longevity, 3.9±0.3 days was observed in combination of LC30 of Ma-2.4 and triazophos, while at LC10 the minimum male longevity 7.1±0.4 days was recorded in combination of LC10 of Ma-2.4 and bifenthrin mixture (Table V).

The combined treatments of fungi and insecticides greatly affected the female longevity of O. hyalinipennis. The results showed that the combined mixture of Ma-2.4 and bifenthrin at LC40, LC30 and LC20 reduced female longevity to 3.1±0.8, 5.0±0.7 and 10.0±0.9 days, respectively. While the mixture of LC10 of Ma-2.4 and triazophos reduced female longevity to 12.6±0.8 days.

There is a great interest all over the world for utilizing and manipulating entomopathogenic fungi for biological control of insects. Entomopathogenic fungi are just like parasite which kill or disable the insects. Unlike other pathogens like bacteria and viruses which need to be ingested for their action these fungi require contact with cuticle under favorable temperature and humidity (Dhaliwal and Koul, 2007). In order to increase their effectiveness, these can be mixed with insecticides (Pachamuthu and Kamble, 2000). These fungi and selective insecticides may synergize the effect of each other thus increasing the efficiency of control, preservation of natural enemies, reducing insecticides usage, environmental risks and resistance in pests (Quintela and McCoy, 1998; Dayakar et al., 2000; Ambethgar, 2003; Rachapa et al., 2007). Combination of imidacloprid and fungal pathogen showed the synergistic effect for the control of insect pests (Kaakeh et al., 1997; Quintela and McCoy, 1998; Ramakrishnan et al., 1999; Lacey et al., 1999; Ying et al., 2003).

The findings of current study depicted that the binary mixtures of fungi and insecticides at higher doses showed higher mortality than alone application. Highest percent mortality i.e., 90.0 was caused by the mixture of isolate Bb-01 and bifenthrin. It confirms the findings of Purwar and Sachan (2006) that the mixture B. bassiana and endosulfan proved to be more toxic and caused higher mortality in Spilarctia oblique (Walker). Identical results were reported by Dayakar et al. (2000) when combined mixture of B. bassiana and insecticide was applied to Spodoptera litura (Fabricius). B. bassiana is highly compatible with avermectin and pyrethroids than any other insecticide (De Olivera and Neves, 2004). Synergistic effect of B. bassiana and imidacloprid, M. anisopliae and fenitrothion or teflubenzuron were recorded against termite Reticulitermes flavipes (Kollar) and scarab beetle larvae (Boucias et al., 1996; Hornbostel et al., 2005). Previous studies focused on the potential use of combination of insecticides and fungi (Pachamuthu and Kamble, 2000; Zurek et al., 2002; Jaramillo et al., 2005; Thompson et al., 2006; Ericsson et al., 2007; Sharififard et al., 2011). In addition to the direct effect on insect mortality, the current study also studied the effects on the biological parameters including adult emergence and adult longevity. Binary mixture of entomopathogenic fungi and insecticides greatly reduced the percent adult emergence and adult longevity at higher doses while on other doses there was significant difference in the percent adult emergence and adult longevity than normal which is in accordance to study of Ekesi et al. (2002) which showed low adult emergence in S. litura when treated with fungus. The results of current research are in accordance to the findings of Pelizza et al. (2013), who validated the decrease in the survival rate and lower fecundity in dipterans when treated with the EPF. The present research shows that the mixture of pathogenic fungi and insecticides increased the management; however, more work is needed to study the compatibility of these fungi and insecticides under field conditions.

Table VI.- Female longevity of O. hyalinipennis after the treatment of binary mixture of entomopathogenic fungi and insecticides.

The present research shows that the mixture of pathogenic fungi and insecticides increased the management by increasing mortality and decreasing the development time of different stages of O. hyalinipennis; however, more work is needed to study the compatibility of these fungi and insecticides under field conditions.

The authors declare no conflict of interest.