INTRODUCTION

Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) is one of the key limiting factors of maize crop due to its efficient colonizing behavior in different agro ecological zones of the world (Tamiru et al., 2007). In the last few years C. partellus has become a major devastating pest of not only maize crop but also of sorghum, causing heavy yield losses and hence requires effective management (Asmare et al., 2011; Tamiru et al., 2012).

The larval feeding results in the destruction of the shoot apex, stem breakage and lodging, disruption of nutrient translocation, stunting and direct damage to tasseling as well as ears (Kfir et al., 2002). The damage potential of C. partellus varies with altitude, moisture gradient, plant species, season, geographical areas, stage of plant growth and plant protection practices (Songa et al., 2002; Ongamo et al., 2006). In cases of severe attack of this pest at cob-formation and grain-filling stage, the infesting larvae damages the grains and ultimately reduces yield to a great extent (Bergvinsion et al., 2004; Mashwani et al., 2015). A substantial loss of 20-25% by C. partellus has been reported in Kenya in dry transitional and lowland tropics (Ongamo et al., 2006). Moreover, Farid et al. (2007) reported 8-20%, 10-22%, 10-21%, 9-22% and 8-22% damages on vegetative, grain-filling, earing, silking and tasseling stages of maize crop, respectively in Peshawar valley of Pakistan. Depending upon the type and nature of the crop, 60 and 80% losses occurred in grain-maize and fodder-maize, respectively while 88% losses in sorghum have been documented due to C. partellus infestation (Jalali & Singh, 2003).

Traditionally farmers rely on the use of chemical insecticides for the control of C. partellus particularly granular formulations (Carbofuran 3G) applied as a whorl treatment. Chilo partellus control using only one method such as chemical control has proved to be a difficult proposal due to several limitations. The predominant use of chemicals has resulted in resistant populations of the pest, resurgence of minor pests, destruction of beneficial insects and buildup of insecticidal residues in the environment (Bruce et al., 2010). Moreover, effective chemical control has been hampered due to the high costs involved and inefficacy due to the pest´s cryptic feeding behavior (Reddy et al., 2009).

The increased pest selectivity, low risk to elicit insect resistance and environmental safety makes entomopathogenic microorganisms a suitable alternative of commercially available chemical insecticides (Gao et al., 2017). They can be used either singly or in combination with other pest management techniques without affecting beneficial arthropods (Hernandez-Rosas et al., 2020). The spore forming entomopathogenic bacterium Bacillus thuringiensis (Bt) (Berliner) and the fungus Metarhizium anisopliae (Mechnikov) play a valuable part in different crop protection approaches with ecologically sound and high levels of efficacy (Zimmermann, 2007).

The fungal isolates of M. anisopliae have been a valuable asset in modern pest control tactics because of their unique contact action mechanism and ability to distribute fungal inoculum through secondary infection (Shah & Pell, 2003). Upon fungal contact, the cuticle of the host insect is degraded through the production of different enzymes which further facilitate the fungal infection (Shan & Feng, 2010; Ramanujam et al., 2011). The fungal spores produced inside the insects body ultimately diminishes feeding, reproductive potential, development and various physiological processes leading to death (Scholte et al., 2003; Mascarin et al., 2019). Bacillus thuringiensis (Bt), is a target specific biological control agent with unique insecticidal properties possessing no detrimental effects on beneficial insects and environment (Plata-Rueda et al., 2020). The cry toxins produced by B. thuringiensis during its growth phases, targets the host upon ingestion (Kumar, 2003). Toxin exposure often results in feeding interruption due to paralysis of the gut and mouthparts along with the formation of pores in the apical microvilli membrane cells, leading to sepsis (Bravo et al., 2005). The specific action of these toxins against coleopteran, dipteran and lepidopteran pests commends their application on different grain crops where injudicious use of chemical insecticides cause severe ill effects (Van Frankenhuyzen, 2009). Apart from their direct action against target pests, a score of fungal entomopathogens such as M. anisopliae been reported to grow within plant cells, paving the way for their potential use as endophytes in pest regulation strategies (Ambele et al., 2020).

Moreover, an increased efficacy of both entomopathogens against lepidopteran pests have also been reported when used simultaneously (Gao et al., 2012; Wakil et al., 2013). The increased synergistic effect of both entomopathogens is mainly attributed to nutritional arrest of insects when exposed to Bt making them more vulnerable to fungal infection (Kryukov et al., 2009). Keeping in mind the significance of these entomopathogens in pest control programs, the present trial was executed to explore the individual and combined effects of M. anisopliae and B. thuringiensis on the mortality, pupation, adult emergence, mycosis and sporulation of 2^nd and 4^th instar larvae of C. partellus.

MATERIALS AND METHODS

Insect culture

Different larval instars of C. partellus were collected from maize and sorghum fields with the permission of farmers which were previously not exposed to any pesticidal or entomopathogen treatments. The collected larvae were placed in plastic jars and taken to the Microbial Control Laboratory (MCL), Department of Entomology, University of Agriculture, Faisalabad, Pakistan. The collected larvae were reared individually on artificial diet (Kfir, 1992) a six-well plate to avoid cannibalism. The laboratory conditions were maintained at 26±2 °C, 75±5 RH and 12:12 D: L. The emerging pupae were transferred immediately into perforated rectangular plastic boxes (28 × 17 × 18 cm.). The newly emerged adult moths were recorded, sexed daily and transferred to plastic jars (15 cm in diameter × 19 cm in depth) lined with wax paper as nappy liner for egg laying. Two percent formaldehyde solution was used for initial sterilization of collected eggs followed by washing and rinsing with tap and distilled water respectively. The eggs were then placed on paper towels for air-drying and kept in plastic bags for maintaining the next progeny (Marzban et al., 2009). The adults were provided with 10% honey solution using a cotton swab placed vertically on the top of the jar in a 5-ml test tube. Newly emerged larvae of 5^th generation were used for experimental trials.

Entomopathogenic fungi

The fungal strain of M. anisopliae (WG-44) (Yasin et al., 2019) used in the current trial was obtained from the culture collection of MCL, originally isolated from infected cadavers of Rhynchophorus ferrugineus (Olivier) by single spore method (Choi et al., 1999) and cultured on Sabouraud dextrose agar (SDA) plates kept at 25±2 °C and 75% RH in the dark. Two to three weeks old sporulated culture was used for harvesting conidia which were later suspended in 10 mL of distilled water with 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) in universal bottles containing glass beads (6-9 beads 3 mm in diameter per bottle). To break the conidial clumps to ensure a homogeneous mixture, the fungal suspension was vortexed at 700 rpm for five minutes. After this, the spore suspension was filtered through four layers of sterile cheesecloth in order to remove residues of mycelium and agar pieces. The desired conidial concentrations (1×10⁴, 1×10⁶ and 1×10⁸ conidia/ml) were verified using a Neubauer improved haemocytometer (Marienfeld, Germany).

Preparation of B. thuringiensis spore‑crystal mixtures

Similarly, Dipel®, a commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was provided by Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering and Biotechnology, Thailand. Sporulation of the Bt strain was done by culturing it in 50 ml nutrient broth media which was latterly subjected to centrifugation for 15 minutes at 6000 rpm. The pellets formed were re-suspended after three times washing with cold 1 M NaCl solution. The adjustment in spore concentration was done at 1:100 dilutions while measurement of optical density was done at 600 nm (Hernández et al., 2005). The samples were then stored in refrigerator (4 ºC) until used (Wakil et al., 2013).

Bioassay

For individual applications, newly emerged 2^nd and 4^th larval instars of C. partellus were subjected to fungal doses (1×10⁴, 1×10⁶ and 1×10⁸ conidia/ml) for 10 s through larval immersion method. After fungal treatments, larvae were air-dried for 10 minutes in sterile Petri dishes and shifted to six-well plates provided with artificial diet (Kfir, 1992) until pupation. The rearing conditions were maintained at 26±2 °C, 75±5 RH and 12:12 (D: L). Similarly, B. thuringiensis (0.75 µg/g) was offered to both larval instars through diet. Larvae were reared on Bt incorporated diet until pupation. For determining the integrated application potential of both entomopathogens, larvae were first dipped in fungal concentrations and later provided with Bt amended diet until pupation. However, control larvae were dipped into a suspension of 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) without any fungal spores and later offered Bt-free diet. All the treatments were arranged in completely randomized design (CRD) using 25 larvae /replicate. The whole experiment was repeated nine times to calculate the percent mortality, percent pupation and percent adult emergence of maize stem borer.

Mycosis and sporulationMycosis and sporulation

For mycosis and sporulation, the cadavers of C. partellus from each treatment were collected and refrigerated at 4 °C in plastic vials. All collected cadavers were sterilized with sodium hypochlorite solution (0.05%) for 2-3 minutes. After, sterilization the cadavers were rinsed three times with distilled water, placed on SDA plates and incubated at 25±2 °C and 75% RH for 7-9 days. After 7 days of incubation mycosed insects were isolated by examining fungal growth on cadavers under the stereo microscope. The data for sporulation were recorded by mixing mycosed cadavers from each replication in 20 ml distilled water with a drop of Tween-80 (Riasat et al., 2011). The solution was thoroughly stirred in such a way that all the spores detached from the insects’ body. The stirring was done until a homogeneous solution was ensured and total number of conidia/ml were counted with the help of a haemocytometer under the microscope.

Statistical analysis

The mortality in control treatments was less than 5%, so treatment mortalities were corrected using Abbott’s formula [Corrected mortality (%) = (1- insect population in treated unit after treatment/insect population in control unit after treatment) × 100] (Abbott, 1925). For each larval instar, mortality values were subjected to a one-way analysis of variance (ANOVA). The same analysis was used to obtain the means for pupation, adult emergence, mycosis, and sporulation. All the analyses were conducted with MINITAB 13.2 statistical package (Steel et al., 1997). The Tukey-Kramer (HSD) test at the 5% significance level (Sokal & Rohlf, 1995) was used to compare the means of larval mortality, pupation, adult emergences, mycosis and sporulation values.

RESULTS

Larval mortality

A significantly higher (p ≤ 0.05) mortality of both larval instars of C. partellus was recorded at higher fungal concentrations and combined application of both entomopathogens. Maximum mortality in 2^nd (94.63%) and 4^th (91.43%) larval instar was recorded when they were subjected to highest dose rates of M. anisopliae + Bt (Ma3 +Bt: 1×10⁸ conidia/ml +0.75 µg/g) (Table I).

Maximum mortality in individual application of both entomopathogens was recorded in case of Ma3 (1×10⁸ conidia/ml) (2^nd: 53.03% and 4^th: 45.34%) followed by B. thuringiensis (Bt) 0.75 µg/g) (2^nd: 40.37% and 4^th: 34.61%). However, larval mortality gradually declined in both individual and combined entomopathogen treatments at lower fungal concentrations. The results further showed that the susceptibility of 2^nd instar larvae was more pronounced in all tested treatments of M. anisopliae and B. thuringiensis than 4^th instar larvae (Table I).

Pupation and adult emergence

The treatments with both entomopathogens showed significant variations (p ≤ 0.05) regarding of pupation and adult emergence of 2^nd and 4^th larval instar of C. partellus compared to the control. Larval metamorphosis into pupae and adults was significantly lower in treatments where mortality rate was high (Table I).

A greater decline in pupation (2^nd: 0.00% and 4^th: 6.66%) and adult emergence (2^nd: 0.00% and 4^th: 0.00%) of both larval instars of C. partellus was observed in case of combined application of highest dose rate of M. anisopliae (Ma3: 1×10⁸ conidia/ml) and B. thuringiensis (Bt) 0.75 µg/g). The results showed that besides untreated check, minimum pupation and adult emergence of 2^nd (73.33% and 65.71%) and 4^th (82.78% and 70.92%) instar larvae of C. partellus was observed in treatments, where larvae were subjected to individual application of lowest dose rate of M. anisopliae (Ma¹: 1×10⁴ conidia/ml) (Table I).

Mycosis and sporulation

Mycosis and sporulation in cadavers of 2nd and 4th larval instars of C. partellus varied significantly (p ≤ 0.05) against the tested conidial concentrations of M. anisopliae applied either singly or in combination with B. thuringiensis (Fig. 1, 2).

Maximum mycosis and sporulation in 2^nd (88.52% and 143.96 conidia/ml) and 4^th (82.04% and 131.85 conidia/ml) larval instars were noted in case of the lowest individual concentration of M. anisopliae (Ma¹: 1×10⁴ conidia/ml). However, mycosis and sporulation in cadavers of both larval instars of C. partellus gradually declined at higher conidial concentrations (Figs. 1, 2), whereas minimum mycosis and sporulation in cadavers of 2^nd (19.88% and 87.59 conidia/ml) and 4th (14.19% and 69.67 conidia/ml) larval instars was recorded form treatments where higher fungal concentration of M. anisopliae (Ma³: 1×10⁸ conidia/ml) was applied in combination with B. thuringiensis (Bt: 0.75 µg/g) (Figs. 1, 2).

DISCUSSION

Chemical insecticides remain the mainstay of farmers for controlling notorious field and greenhouse pests. However, the efficacy of these synthetic chemicals has been compromised due to their hazardous effects on environment and non-target organisms, emergence of multiple resistant insect biotypes and cryptic feeding nature of insect pests (Hussain et al., 2013). Alternatively, microbial agents particularly entomopathogenic fungi and bacteria possess the required potential to control these cryptic insects due to their unique action mechanism (Wakil et al., 2017). Moreover, these agents also pose negligible ill effect on environment as compared to synthetic chemicals (Faria & Wraight, 2007).

The present research was aimed to evaluate the effectiveness of M. anisopliae and B. thuringiensis indicating that M. anisopliae resulted in significant mortality (> 40%) at maximum dose rate in either larval instars, especially 2^nd instar where mortality was more than 50%. This may be attributed to the fact that fungal spores may play a key role in suppressing the neonates of maize stem borer feeding externally on maize leaves however; the older instars may not be infected to the same extent by the fungal application possibly due to their cryptic feeding behaviour inside the stem (Tefera & Pringle, 2004). The current study also revealed similar mortality levels in both larval instars of C. partellus where higher mortality was recorded at higher conidial concentrations as compared to lower doses. The pathogenic potential of M. anisopliae against neonates of Helicoverpa armigera (Hübner) was also reported by Nguyen et al. (2007). Our results are also consistent with the findings of Sasidharan & Varma (2005), who reported that the mortality of Indarbela quadrinotata (Walker) (6 weeks old larvae) increased from 66.7% to 100% when subjected to higher fungal concentrations.

The susceptibility of different developmental stages of insect pest vary when subjected to fungal treatments (Inglis et al., 2001). The present findings also demonstrated that vulnerability of 2^nd larval instar of C. partellus was more evident in all tested treatments than its 4^th instar larvae. The possible reason could be the larval instar; the body of early instar larvae is soft and immune system is not well developed, whereas later instar larvae become more resistant to fungal infection and its immune system is well developed compared to the neonates. The observed lower mortality rates in later instar larvae could be related to decline in penetration of fungal germ tube inside insect's body due to increased melanin levels in the insect's midgut and cuticle (Wilson et al., 2001) or cryptic feeding behavior of later instar larvae that are infected with the secondary infection from mycosed cadavers (Tefera & Pringle, 2004). Similar results were also reported by Hafez et al. (1997), who discovered that Beauveria bassiana (Balls) Vuill. was more virulent to early larval instars of Phthorimaea operculella (Seller) as compared to its latter instars. Similarly, Sufyan et al. (2019) also reported higher susceptibility of 2^nd instar larvae of C. partellus then its 4^th larval instar against B. bassiana.

Likewise the virulence of Bt was also found to have an inverse relation with C. partellus larvae age. The reduction in efficacy of Bt against older larval instars of Helicoverpa zea (Boddie) was also reported by Herbert & Harper (1985). Similarly, 2^nd and 3^rd instar of Colorado potato beetle suffered 98% and 52% mortality respectively, when both instars were subjected to similar toxin levels (Zehnder & Gelernter, 1989). Moreover, Lacey et al. (1999) observed that the administration of low to high quantities of B. thuringiensis provided intermediate to effective management of Colorado potato beetle. Variations in mortality among different larval instars exposed to similar treatments might be due to differences in enzymatic activities. It has been found that the activity of detoxification enzymes varies considerably across and within different growth stages of insects. This effect is usually minimal at the egg stage, increases with every larval stage, and eventually decreases to zero during the pupal phase (Ahmad, 1986; Mullin, 1988).

Our results also suggest that combined applications of both entomopathogens caused higher larval mortality in both instars of C. partellus. These results are consistent with those of Opisa et al. (2018) who reported pathogenicity of different isolates of M. anisopliae, B. bassiana and lsaria fumosorosea (Wize) against Spoladea recurvalis (Fabricius), with M. anisopliae ICIPE 30 and B. bassiana ICIPE 725 causing the highest mortality of 92% and 83%, respectively. Similarly, an increased larval mortality of Ostrinia nubilalis (Hübner)and Leptinotarsa decemlineata (Say)was also reported due to combined applications of B. bassiana and B. thuringiensis (Lewis et al., 1996; Wraight & Ramos, 2005). Similarly, Sufyan et al. (2019) also reported higher larval mortality of C. partellus when they were exposed to combined applications of B. bassiana and B. thuringiensis. The synergistic action mechanism of entomopathogenic fungi and bacteria was further substantiated by Gao et al. (2012), who discovered that food stress induced by Bt intoxication might induce detrimental impacts on the hosts´ immunity and physiology. This halted nourishment caused by Bt (Kryukov et al., 2009) rendered the host more susceptible to fungal spores, which further accelerated the death process. The starvation stress also increased inter-molt period of insects making them vulnerable to different control tactics for a longer time frame (Furlong & Groden, 2003; Lawo et al., 2008).

The present findings also revealed that mycosis and sporulation in cadavers of both larval instars of C. partellus was much higher at lower conidial concentrations. Mycosis and sporulation in dead insects rely on temperature, application method and conidial concentrations. Larvae die rapidly at higher fungal concentrations thus sporulation process slows down due to limited nutrients in dead insects. Moreover, at higher fungal concentrations, a self-inhibiting between fungal spores was also reported by Tefera & Pringle (2003). Similar self-inhibiting mechanism had also been described for other fungal species against different host cadavers (Garraway & Evans, 1984; Riasat et al., 2011). Likewise chalkbrood fungus, Ascosphaera aggregata Skou has a higher sporulation rate on cadavers of Megachile rotundata (Fabricius) after treatment of intermediate concentrations of fungi as compared to its higher concentrations (Vandenberg, 1992). Similar trend of mycosis and sporulation at lower conidial concentrations had also been described for Rhyzopertha dominica (Fabricius), C. partellus and R. ferrugineus (Riasat et al., 2011; Sufyan et al., 2019; Yasin et al., 2019).

CONCLUSION

The current study has revealed that M. anisopliae and B. thuringiensis are effective against maize stem borer and can be considered as a potential alternative to the use of synthetic chemicals under field conditions. The broader host range of these entomopathogens could also help in controlling multiple insects with a single application. Moreover, they could also provide crop protection for a longer time frame primarily through secondary infection from mycosed cadavers. However, further field research is needed in this regard in order to identify additional virulent entomopathogen isolates, their optimal dosages and better application techniques for effective pest control.

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Notas de autor

asimabbasi@kum.edu.pk