Research Article

A laboratory evaluation of South African strains of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin against adults of Lasioderma serricorne (Coleoptera: Anobiidae) and Sitophilus zeamais (Coleoptera: Curculionidae)

Mohamed Baha Saeed^1,2,3* and Mark D Laing¹

¹Discipline of Plant Pathology, School of Agricultural, Earth and Environmental Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa

²Department of Crop Protection, Faculty of Agriculture, University of Khartoum, Khartoum 11115, Sudan

²Center of Excellence for Pesticides and Plant Health, Faculty of Agriculture, University of Khartoum, Khartoum 11115, Sudan

Submission: March 21, 2024; Published: April 02, 2024

*Corresponding author: Mohamed Baha Saeed, Discipline of Plant Pathology, School of Agricultural, Earth and Environmental Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa, Department of Crop Protection, Faculty of Agriculture, Center of Excellence for Pesticides and Plant Health, Faculty of Agriculture, University of Khartoum, Khartoum 11115, Sudan

How to cite this article: Mohamed Baha Saeed* and Mark D Laing. A laboratory evaluation of South African strains of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin against adults of Lasioderma serricorne (Coleoptera: Anobiidae) and Sitophilus zeamais (Coleoptera: Curculionidae). Agri Res& Tech: Open Access J. 2024; 28(3): 556408. DOI: 10.19080/ARTOAJ.2024.28.556408

Abstract

A total of 21 South African strains of Beauveria bassiana were evaluated for their virulence against adults of Lasioderma serricorne and Sitophilus zeamais, under laboratory conditions. In the first bioassay, strains were applied at a single dose of 1x108 conidia ml-1. Adults of L. serricorne was more susceptible than those of S. zeamais, with 14 strains causing mortality levels > 50.0% on L. serricorne, compared to two strains in the case of S. zeamais. Six strains that caused mortality levels > 90.0% on L. serricorne were compared in a dose-response assay using a water suspension and a powder formulation, with cornflour as the carrier, using five doses (2x104, 2x105, 2x106, 2x107 and 2x108 conidia per volume basis in water (ml-1), and weight basis in the powder formulation (g-1)). The water suspension consistently provided a higher mortality than the powder formulation at all concentrations of conidia. 100% mortality was recorded with a water suspension of 2x108 conidia ml-1 with Strains 7284, 7769 and 7320. With the powder formulation, the highest mortality of 72.2% was recorded at a dose of 2x108 conidia g-1 of cornflour with Strain 7284. In both assays, Strains 7284 and 7769 outperformed the other strains at low doses. These two strains had the lowest LD50 values of 7x104 and 1x105 conidia ml-1 and the shortest lethal times (LT50) of 2.67 and 2.94d with the water suspension, respectively. The result was similar with the powder formulation but there was not much difference between strains. Strains 7284 and 7769 were highly virulent on L. serricorne. However, none of the strains were adequately effective against S. zeamais.

Keywords: Beauveria bassiana; Lasioderma serricorne; Sitophilus zeamais; Biocontrol

Introduction

The maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), is a pest of economic importance in stored products worldwide, particularly of maize (Zea mays L.) in tropical and sub-tropical regions Throne [1]. The pest is capable of multiplying to large populations resulting in severe damage to grains in storage Cosmas et al. [2]. An estimated 40.0% of stored maize is lost to S. zeamais in Africa Meikle et al. [3], and with severe infestations, maize weevils can cause losses of 90.0% Giga et al. [4]. The cigarette beetle (Lasioderma serricorne Fabricius (Coleoptera: Anobiidae)) is found globally in tropical and subtropical areas. This pest is polyphagous, being able to feed on various food sources such as grains, spices, and tobacco Mahroof & Phillips [5].

Fumigants and synthetic insecticides have been used to control pests in grain stores. However, the repeated use of these materials has led to various problems, including insecticide resistance, environmental and human health concerns, mortality of nontarget organisms, and chemical residues in foodstuffs Cherry et al. [6]. Therefore, alternative control strategies are being evaluated, including the use of entomopathogenic fungi. Differences in pathogenicity (virulence) among strains against stored product insects have been reported previously in assays with Beauveria bassiana (Balsamo) Vuillemin and other entomopathogenic fungi on S. zeamais Rondelli et al. [7]; Agoligan et al. [8]; Saeed & Laing [9], Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) Golshan et al. [10]; Golshan et al. [11], Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae) da Paz Júnior et al. [12], Sitophilus oryzae Linnaeus (Coleoptera: Curculionidae) Kavallieratos et al. [13], Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae) Jyothi et al. [14], Plodia interpunctella Hubner, Ephestia cautella Walker and E. kuehniella Zeller (Lepidoptera: Pyralidae) Sabbour et al. [15].

One of the most important steps in the development of a myco-insecticide is the selection of highly pathogenic strains Tefera & Pringle [16]. Little research has been conducted into biocontrol of the cigarette beetle, L. serricorne AiYing et al. [17]; Yuan et al. [18]. Therefore, the objectives of this research were to: (i) identify the most pathogenic of 20 novel strains of B. bassiana and one commercial strain (ARC R444) against adults of the cigarette beetle, L. serricorne, and the maize weevil, S. zeamais, in the laboratory; and (ii) to compare the efficacy of six highly pathogenic strains as either a water suspension or a powder formulation against the cigarette beetle L. serricorne.

Material and methods

Insect rearing

The initial population of insects was obtained from the Department of Plant Pathology, School of Agricultural, Earth and Environmental Sciences (SAEES), University of KwaZulu-Natal. L. serricorne was reared on rice grains. S. zeamais was reared on yellow maize grains. The grains were stored at -20oC for one week to eliminate unwanted natural infestation. Approximately ten adults of each insect of mixed sexes were placed in 250 ml glass jars, each containing 100g of either grain. The jars were covered with insect nets to facilitate air circulation. The adults were removed from the jars after one week of infestation. Two-day-old adults from each insect were used in the experiments.

Fungi

A total of 21 strains of B. bassiana from diverse geographical origins (Table 1) were screened against adults of the cigarette beetle, (L. serricorne) and the maize weevil, (S. zeamais). Twenty strains of B. bassiana were provided by the Plant Protection Research Institute, Agricultural Research Council (PPRI-ARC, South Africa) and the commercial strain of B. bassiana was provided by Plant Health Products (Pty) Ltd (PHP).

Production of conidial suspension

The B. bassiana strains were cultured on potato dextrose agar (PDA) (Biolab Merck (Pty) Ltd. The PDA was prepared using 4g potato extract, 20g dextrose and 15g agar in 1L of distilled water, as directed by the manufacturer, in 9cm diameter Petri dishes and incubated at 28oC for 15 days for complete sporulation. The contents (hyphae and conidia) were harvested by flooding the Petri dishes with 15ml distilled water with 0.01 (v/v) Tween 80 and stirring with a glass rod. The adjuvant Tween 80 was added to facilitate the suspension of conidia in distilled water. Samples were vortexed for 3 minutes to split up conidial clumps. Conidia were separated from hyphae by filtration through three layers of cheese cloth. Conidial concentration was determined using a Neubauer Improved Haemocytometer.

Conidial viability

To assess conidial viability, 0.1ml of each sample suspension was pipetted and thinly spread over PDA in Petri dishes using an “L” shaped glass rod and incubated at 280C for 24h, with three Petri dishes per sample suspension. Conidia were examined at 400x magnification under a light microscope. Germination of a conidia was counted when its germ tube was apparent. All the conidia in each field of view were counted and the percentage germination was calculated. All the strains used in the experiments displayed > 90.0% viable conidia.

First screening bioassay

All 21 strains of B. bassiana were used in the first screening against adults of L. serricorne and S. zeamais in single-dose bioassays under laboratory conditions at 1x108 conidia ml-1. Thirty ˂ two-day old adults of each insect were treated by immersion for 10 seconds in 5 ml of conidial suspension. Each treatment was replicated three times. Control insects were treated with sterile distilled water with 0.01v/v Tween 80. The treated insects were moved to a plate containing filter paper. The filter paper helped to absorb surplus moisture Adane et al. [19]. After 24h the treated insects were transferred into 250ml glass jars with 50g of rice and kept at 28±2oC and 65±5.0% RH for 10 days. The mortality was counted every two days. Dead insects from each treatment were washed in 70.0% sodium hypochlorite, rinsed in sterile distilled water three times and kept in Petri dishes with wet filter paper at 28±oC to observe fungus outgrowth. The experiment was a 22 x 2 factorial, arranged in a randomized complete block design (RCBD). The bioassay was repeated twice. Percentage mortalities of tested insects were corrected relative to the control using Abbott’s formula Abbott [20]. Data was subjected to analysis of variance (ANOVA) using GenStat for Windows, 17th edition Payne et al. [21]. Means were compared using Fisher’s Least Significant Difference test at a 5.0% level of significance.

Production of dry conidia

A concentration of 108 conidia ml-1 was determined using a Neubauer Improved Haemocytometer. Petri dishes with PDA were inoculated by 100μl of fungal suspension and plates were sealed by Parafilm to maintain an adequate moisture level for fungal growth. The inoculated Petri dishes were incubated at 25oC in the dark for 15 days. The Parafilm was then removed from the plates and they were allowed to dry under a laminar flow for 5 days. Conidia were harvested by scraping them off from the surface of the dried medium using a sterile scalpel blade. The harvested fungal tissues were passed twice through a 100 mm diameter sieve (110 μm pore size) to obtain pure powdered conidia. The conidia were stored in sterile sealed bottles at 4oC for further use.

Multiple bioassays using water suspensions and powder formulations

Six strains selected from the first screening bioassay were used in the second bioassay against adults of L. serricorne. Five different doses (2x104, 2x105, 2x106, 2x107 and 2x108 conidia ml-1) of conidial suspension in 0.01 Tween 80 aqueous solutions were prepared for each strain. Each dose was replicated three times. For each replicate, thirty ˂ two days old adults were treated by the immersion method as described in Section 2.2.5. The control treatment was sterile distilled water with Tween 80 (0.01% v/v).

For the powder formulation, conidial doses for each strain were prepared from an initial stock of 2x109 conidia g-1, which was measured using a Neubauer Improved Haemocytometer, and diluted with cornflour to 2x108, 2x107, 2x106, 2x105 and 2x104 conidia g-1. Each dose was replicated three times. For each replicate, thirty ˂ two-day old adults of L. serricorne were introduced into Petri dishes containing 0.01g of the various conidial doses. A control contained the carrier only. After 1 hour the treated insects were transferred into 250ml glass jars with 50g of rice grains and kept at 28±2oC and 65±5.0% RH for 10 days. Mortality was counted every two days for ten days. Glass jars were placed as per a 6 x 6 x 2 factorial experiment arranged in a randomized complete block design (RCBD). This bioassay was performed twice. Mortality was monitored every two days for ten days, and corrected on the basis of natural mortality observed in the control treatment using Abbott’s formula Abbott [20]. Data was subjected to analysis of variance (ANOVA). Means were compared using Fisher’s Least Significant Difference test at a 5.0% level of significance. Probit analysis was used to determine the median lethal dose (LD50) and median lethal time (LT50). All these analyses were performed using GenStat for Windows, 17th edition Payne et al. [21].

Results

First screening bioassay

There were significant differences between fungal strains in their virulence on the two insects (F=64.04; P˂0.001), and the mortality levels that they caused (F= 738.75; P˂0.001). The interaction between fungal strains and insects (F=12.19; P˂0.001) was also significant. All B. bassiana strains were pathogenic and mycelial growth demonstrated that most of the tested insects died due to the fungus (Figure 1). L. serricorne was more susceptible than S. zeamais after 10 days of exposure to the 21 strains of B. bassiana. Mortality varied as a result of the strains, and the mortality levels ranged from 14.29 to 96.4% and 10.71 to 82.20%, for L. serricorne and S. zeamais, respectively (Figure 2). Among the tested strains, six strains (7284, 7320, 7768, 7288, 7769 and R444) were the most virulent against L. serricorne, with mortality levels > 90.0%; eight strains were moderately virulent, inducing 50.0% to 82.2% mortality, and the remaining seven strains caused mortality ˂ 50.0% (Figure 2). Adults of S. zeamais were relatively resistant to B. bassiana, with 20 of the strains causing mortality levels of ˂ 51.0%. However, Strain 7769 was more pathogenic on this insect, and caused an 82.2% mortality when applied at a dose of 1x108 conidia ml-1. The commercial Strain R444 used in this bioassay caused ˂50.0% mortality against S. zeamais, but it was highly virulent on L. serricorne and caused a 92.9% mortality level (Figure 2.2).

Multiple bioassays using water suspensions and powder formulations

All main effects as well as related interactions were highly significant at p˂ 0.001. The dose response for the water suspensions was linear between 2x104 -2x108 with the R2 > 0.94. The same occurred with the powder formulations, with the R2 > 0.95 (Figure 3). Dose responses always reach a plateau where increased doses do not increase mortality. In this experiment, the plateau was reached at a dose of 2x108 conidia with both the water suspensions per ml or the powder formulations per g. Therefore, a useful practical range is 2x104 - 2x108 conidia ml-1, or conidia g-1 ,which is achievable in practice.

Water suspensions of the six selected strains in the first bioassay against the adults of L. serricorne consistently caused higher mortality levels at all doses than the powder formulations. 100% mortality was achieved with a water suspension at 2x108 conidia ml-1 with Strains 7284, 7769 and 7320, compared to a high of only 72.2% achieved with a powder formulation at 2x108 conidia g-1 using Strain 7284. Using water suspensions, Strains 7204 and 7769 caused higher mortalities at low doses than the other strains, which is important because it is at low doses that successful control in granaries will need to occur. The same two strains were also the most virulent at low doses with the powder formulations (Figure 3). The commercial strain R444 used in this study caused the lowest mortality level of 58.7% against L. serricorne, together with Strain 7768, using powder formulations at 2x108 conidia g-1. However, R444 caused a higher mortality (96.5%) against the same insect when used in a water suspension at 2x108 conidia ml-1 (Figure 3).

Probit analyses were performed separately for each strain formulated in a water suspension or powder formulation, and the results are presented in Figure 4 & Figure 5. The 21 B. bassiana strains in water suspensions achieved lower LD50 and LT50 values than they did in the powder formulations.

Using a water suspension, Strain 7284 achieved the lowest LD50 value (log (LD50 = 4.849)) and the shortest LT50 value (LT50 = 2.69 days) followed by Strain 7769 (Log (LD50 = 5.019) and (LT50 = 2.94). The results were similar for powder formulations, but the differences between strains were reduced. The six strains performed relatively well, and even the least virulent strain caused significant mortality levels.

Discussion

The present study evaluated the pathogenicity of 21 strains of B. bassiana (including a commercial strain (R444)) applied to adults of L. serricorne and S. zeamais, with a 10 day of exposure period. Although all the tested strains were pathogenic and caused some mortality of the tested insects, there was a wide range in the levels of virulence. The cause of such variation in virulence can be attributed to differential virulence by the strains, or by differential resistance by the host insects. All the cadavers of treated insects showed outgrowth of fungal mycelium, confirming that the fungus was the main cause of insect death, none being detected in cadavers in the control treatments.

L. serricorne was more susceptible than S. zeamais to the tested strains. Among the tested strains, 14 strains achieved more than 50.0% mortality levels against L. serricorne, with only seven recording ˂ 50.0% mortality. These results are similar to those of Cherry et al. [22] who found that 100% mortality of C. maculatus resulted from exposure to the aqueous suspension of various strains of B. bassiana and M. anisopliae at a doses of 1x108 conidia ml-1 at six and eight days after treatment, respectively.

In contrast, S. zeamais was relatively resistant to the 21 strains of B. bassiana used in this study, with 20 strains achieving mortality levels ˂ 51.0%, and one strain causing 82.2% mortality. Shams et al. [23] observed a similar pattern with S. granarius , which was less susceptible than C. maculatus following exposure to five different conidial concentrations of B. bassiana under laboratory conditions. In the same context Rondelli et al. [7] reported that all twelve isolates of B. bassiana tested against S. zeamais adults caused pathogenicity. However, there were highly significant differences among the isolates with respect to virulence, with corrected mortality levels ranging between 19.7 and 72.0%, but only three of the twelve isolates caused mortality levels > 50.0%. Golshan et al. [10] used nine isolates of B. bassiana against the adults of T. castaneum, and these caused mortalities of between 15 and 60.0%, with only two isolates achieving mortality levels > 50.0%.

The six most virulent strains from the first bioassay were compared in dose response assays of a water suspension, and a powder formulation, using cornflour as the carrier, against adults of L. serricorne. The water suspension consistently achieved higher mortality levels at all conidial doses, and lower LD50 and LT50 values than the powder formulations. The high humidity associated with the water suspensions creates a conducive environment for infection by B. bassiana Devi et al. [24]. The higher mortality levels and lower LT50 achieved by Strains 7284 and 7769 at the lower doses are important because biocontrol of storage pests in granaries will need to be achieved at similarly low doses. In addition, 100% mortality was also observed with these strains at 2x108 ml-1. Several authors have also reported that using the aqueous conidial suspensions of B. bassiana and other entomopathogenic fungi increased the efficacy of these fungi toward target insects and resulted in high mortality levels Teshome & Tefera [25]; Mahdneshin et al. [26]; Faraji et al. [27]; Uçaret al. 2020). Although using water suspension achieves high mortality levels, it would not be practical to use these as a product against grain storage pests because the treatment of grains with aqueous suspensions would create high humidity conditions in storage that would promote the emergence of fungal moulds.

Use of a powder formulation at 2x108 g-1 with Strain 7284 resulted in a 72.2% mortality, with higher LD50 and LT50 values than the water suspension. Nevertheless, powder formulations are more practical for the treatment of stored grains. These results are in agreement with Khashaveh et al. [28] who reported mortality levels of 88.4, 78.4 and 65.0% for S. granarius, Oryzaephilus surinamensis Linnaeus (Coleoptera: Silvanidae) and T. castaneum, respectively, following their exposure to wheat grains treated with a dry formulation of B. bassiana (Bb WeevilTM, a commercial product containing 2x109 conidia g-1). This was applied at a concentration of 1000 mg kg-1, equivalent to ten times the conidial concentration of conidia tested in this study. Saeed & Laing (2023) reported on a six-month study in which a formulation of B. bassiana Strain MS-8 applied in kaolin (1x109 conidia g-1) at levels of 1 g kg-1 of grain performed well, resulting in a low number of live weevils (36 insects/500g of maize grain), a low level of grain damage (14.0%), and a low level of weight loss (7.0%). In contrast, in the untreated control, the number of live insects was 340 insects/500g of maize grain, the level of grain damage was 68.0%, and the weight loss was 51.0%.

Conclusion

In conclusion, B. bassiana Strains 7284 and 7769 were the most effective for control of cigarette beetle, L. serricorne, either as water suspensions or powder formulations. However, there is a need to screen for more virulent strains against the maize weevil, S. zeamais, for use in the management of this pest.

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