Establishment of Beauveria bassiana isolates as endophytes in rice cultivars and their biocontrol efficacy against rice stem borer, Sesamia calamistis

Volume 116| Number 11/12 November/December 2020 Research Article https://doi.org/10.17159/sajs.2020/7914 © 2020. The Author(s). Published under a Creative Commons Attribution Licence. Establishment of Beauveria bassiana isolates as endophytes in rice cultivars and their biocontrol efficacy against rice stem borer, Sesamia calamistis AUTHORS: Wonroo B.A. Bancole1 Mark D. Laing1 Kwasi S. Yobo1 Abou Togola2


Introduction
Rice (Oryza spp L.) is one of the world's most important crops, providing food for more than half of the world's population. [1][2][3] Rice and wheat (Triticum spp L.) together contribute about 21% of the total energy consumed by humans. 4 In West Africa it has become the main source of calories for low-income households. 5 Two Oryza spp. are cultivated globally: Asian rice (Oryza sativa L.) and African rice (Oryza glaberrima S.), for which the cultivation is limited to tropical West Africa. 6 Rice is now grown and consumed in more than 40 countries on the African continent. 7 Its consumption has increased rapidly in Africa, making it the second largest source of carbohydrates in sub-Saharan Africa. 4 Imports of rice account for nearly 40% of the total rice consumption of the region. 8,9 Losses caused by biotic factors such as pests, diseases and weeds reduce yields of rice worldwide. According to estimates of the Food and Agriculture Organization of the United Nations (FAO), diseases, insects and weeds cause as much as 25% yield losses annually in cereal crops. 2 The most serious pests of rice plants worldwide are rice stem borers, which belong to three families (Noctuidae, Pyralidae and Diopsidae). Sesamia calamistis Hampson (Lepidoptera: Noctuidae) is one of the major pests that attacks grain crops including rice, maize (Zea mays L.), pearl millet (Pennisetum sp L.), wheat, sorghum (Sorghum bicolor L.) and sugarcane (Saccharum officinarum L.). The control of this borer by commercial farmers has mainly relied on the application of synthetic insecticides. [10][11][12] However, control of S. calamistis using chemicals is difficult because of a prolonged emergence pattern, multiple generations and a cryptic feeding behaviour. 13 A further issue is that, as with many other stem borers, S. calamistis has developed resistance to chemicals. In addition to the insecticides' high costs and their inefficacy against the borers, they may also cause ecological problems, and are usually unaffordable for small-scale farmers. [14][15][16][17] The need for alternative methods for the control of major pests has driven research to develop biological control products. Indigenous predators, parasites and entomopathogens are the most commonly used biological control agents in tropical Asia and Africa to control stem borers. 18 Entomopathogenic fungi are important among biological control agents due to their broad host range, their diverse mechanisms of pathogenicity, and their environmental safeness. [19][20][21] Some strains of the entomopathogen B. bassiana have been introduced into several plant species [maize, banana (Musa spp L.), tomatoes (Solanum lycopersicum L.), sorghum, coffee (Coffea arabica L.), wheat and pumpkins (Cucurbita spp D.)] to control various insects. [22][23][24][25] Various inoculation methods (seed treatments, soil drenches, foliar and flower sprays, and stem injections) have been used for their establishment as endophytes in those crops. The main reason for conducting this study was to determine if endophytic strains of B. bassiana in rice cultivars might provide protection against S. calamistis, the major rice stem borer prevalent in West Africa.

Materials and methods
Five isolates of B. bassiana previously identified as endophytes in sorghum were evaluated for their potential establishment as endophytes in rice cultivars plant tissues. Third larval instars of S. calamistis were used as the test insects.

Production of conidial suspensions of B. bassiana isolates
Five B. bassiana isolates (Bb3, Bb4, Bb10, Bb21 and Bb35) were used for the study. These had been isolated from various soils, including soil samples collected from the rhizosphere of mangoes (Mangifera indica L.), rooibos tea (Aspalathus linearis Burm. f.) and wheat. They were characterised by the Plant Protection Research Institute (Pretoria, South Africa). In prior research, they were selected for their endophytic abilities in sorghum plant tissues (leave, stem and root). 18 Conidial suspensions used for the study were prepared following the method of Parsa et al. 18 The strains were cultured in 90-mm diameter plastic Petri dishes containing potato dextrose agar (PDA) supplemented with antibiotics (100 mg/L of ampicillin and streptomycin), and incubated at 28 °C. The cultures were allowed to grow for 14-18 days, after which the conidia were harvested. The conidia were harvested under sterile conditions by gently scraping the fungal growth from the surface of the agar with a sterile spatula, and rinsing with sterile distilled water. The resulting suspensions were filtered using sterile cheese cloth to remove mycelia and agar debris. Conidial density was determined using an improved Neubauer haemocytometer, and adjusted to 2x10 6 conidia/ mL with sterile distilled water containing Tween-80 (1 mL/L). The viability of the conidia for all the experiments was evaluated by taking a 100-μL sample of each strain, spreading it on PDA and incubating at 25 °C. Conidia germination was assessed after 24 h of incubation. The percentage germination of conidia was determined from 100 randomly selected conidia under a light microscope. The germination of conidia was assumed when the hyphae were visible or the germ tube was about twice the length of the conidium. For each strain, the mean of three replicates was used to assess the viability of the conidia. The final inoculum was used for seed treatment and foliar spray experiments.

Production of rice plants for greenhouse studies
Three African rice cultivars (NERICA1, NERICA8 and NERICA-L19) 26 were used as the host plants as there may be differential interactions between host plants and endophytic strains. Seeds of each cultivar were separately surface sterilised in 3% sodium hypochlorite for 3 min followed by 70% ethanol for 2 min. They were rinsed three times with sterile distilled water, air dried on a laminar flow bench and then divided into two sets. The first set was used for seed inoculation and the second for foliar spray experiments. The second set of seeds used for foliar spray experiment were sown in Speedling ® 24 trays filled with Composted Pine Bark (CPB) seedling mix growing medium. The seeds were watered with tap water and placed under greenhouse conditions at 20-28 °C day and night. Two weeks after germination, the seedlings were transplanted into 30-cm diameter pots filled with CPB seedling mix growing medium and placed under greenhouse conditions at 20-28 °C day and night. The plants were allowed to grow for 7 days before being used in the foliar spray experiment. Plants were irrigated three times a day with irrigation water containing NPK fertiliser [3: 1: 3 (38)] (50%) together with calcium nitrate (50%) and trace elements.

Seed treatment
The seeds of the three rice cultivars were surface sterilised as previously described. After surface sterilisation, the seeds for each cultivar were separately soaked in the conidial suspension of each B. bassiana isolate [5 mL of the prepared inoculum (2×10 6 conidia/mL)], allowed to stand overnight, then removed and air dried on a laminar flow bench. The seeds were then planted in Speedling ® 24 trays filled with CPB seedling mix growing medium. The control plants consisted of non-inoculated seeds treated in a similar manner using sterile distilled water. After 2 weeks, the emerging seedlings were transplanted into 30-cm diameter pots filled with CPB seedling mix growing medium and placed under greenhouse conditions at 20-28 °C day and night. Three plants per pot were arranged in the greenhouse in a randomised complete block (RCB) design with three replicates. Plants were irrigated three times a day with irrigation water containing NPK fertiliser [3: 1: 3 (38)] (50%) together with calcium nitrate (50%) and trace elements. The plants were grown for 30 or 60 days before they were harvested, and the roots, stems and leaves were evaluated for evidence of endophytic colonisation.

Foliar spray
The seedlings of the three rice cultivars were sprayed 15 days after transplanting into pots. A hand spray was used to inoculate the rice plant leaves with the inocula of the B. bassiana isolates. A volume of 50 mL inoculum of each B. bassiana isolate was used per plant. Before the leaves were sprayed, the base of each pot was covered with aluminum foil, with a hole to allow the plant to emerge. This was to stop inoculum running off the leaves onto the roots and creating a root drenching situation. Plastic bags were used to cover the entire plant for 24 h to increase humidity. For the control plants, sterile distilled water was applied in a similar manner as described for the B. bassiana treatments. The treated plants (three plants per pot) were then placed in a greenhouse (20-28 °C day and night) using a RCB design with three replicates. Plants were irrigated three times a day with irrigation water containing NPK fertiliser [3: 1: 3 (38)] (50%) together with calcium nitrate (50%) and trace elements. The roots, stems and leaves of each treated plant were harvested after 30 and 60 days, for evaluation for endophytic colonisation.

Evaluation of endophytic colonisation of the B. bassiana isolates
The colonisation of rice plant tissues by B. bassiana was determined 30 and 60 days after inoculation with each B. bassiana isolate. From each rice cultivar x B. bassiana treatment combination, plants were carefully removed from their pots and sampled into leaves, stems and roots. The roots were gently washed with tap water to remove residues of CPB. The plant tissues were surface sterilised by immersing them in 3% sodium hypochlorite for 3 min, followed by 70% ethanol for 2 min. They were rinsed three times with sterile distilled water. The surface sterilised samples were placed on sterile tissue paper under a laminar flow cabinet for air drying. After drying, six pieces of each of the samples (leaves, stem and roots) from each treated plant were randomly taken and plated separately onto a B. bassiana selective medium (39 g/L PDA +2 g yeast extract + 1.1 g Dodine + 100 mg/L of streptomycin and ampicillin) 27 and incubated for 15 days at 25 °C. To confirm that the surface sterilisation was effective, 10 mL of the sterile distilled water used to rinse the samples during the surface-sterilisation procedure was spread onto Petri dishes containing the B. bassiana selective media. The plates were incubated for 10-15 days at 25 °C to count the colony forming units. However, the sterilisation resulted in clean plates. Therefore, any B. bassiana mycelium emerging from surface-sterilised plant tissues was assumed to have originated from within the plant tissues as an endophyte. The plates that contained the plant samples were monitored every 2-3 days for the emergence of fungal mycelia. After 10-15 days, the presence or absence of B. bassiana colonies were recorded. The fungal colonies grown from the samples were confirmed to be B. bassiana based on morphological characteristics.

Mass rearing of S. calamistis larvae
A suitable number of S. calamistis pupae collected from a maize field were placed into cages that contained cotton soaked in sugar/honey water that served as food for the moths once they emerged from the pupae. Sheets of transparent paper were wound around wooden rods (40 cm) to create a slot for the female moths to lay their eggs. These eggs were harvested from the slots by scraping the wooden rods with a sterile spatula. The eggs were placed into plastic containers on a sterile paper towel. The containers were incubated at 26 °C with a relative humidity of 60% (±10%), and a photoperiod of 12-h light and 12-h dark. The containers were monitored daily until the larvae hatched, creating the stage called 'black heads' (first instars). These young larvae were transferred into other transparent plastic containers whose lids were perforated but covered with mosquito netting to ensure permanent ventilation, and were incubated at 26 °C. Fresh maize stalks were harvested and placed into the plastic containers to serve as food for the black head larvae. After 3 days, the maize stalks were dissected and the larvae were extracted and transferred into new containers that contained fresh maize stalks (Figure 1), and kept in an incubator at 26 °C. This procedure was repeated until larvae of the desired third instar larval stage had developed. The number of days for each stage varied from one larval stage to the next. Development from the second to the third instar took 5 days, while development from the third to the fourth instar took 8-10 days.

Production of endophytic stems of rice plants
Seeds of a rice cultivar (NERICA 1) were surface sterilised in 3% sodium hypochlorite for 3 min, followed by 70% ethanol for 2 min. They were then rinsed three times with sterile distilled water and air dried. The surfacesterilised seeds were then dipped separately in a conidial suspension of each of the five B. bassiana isolates [5 mL of the prepared inocula (2×10 6 conidia/mL)] and left overnight before air drying under a laminar flow cabinet. The seeds were then planted in Speedling ® 24 trays filled with CPB seedling mix growing medium. After 2 weeks, the seedlings were transplanted into 30-cm diameter pots filled with CPB seedling mix growing medium and placed under controlled greenhouse conditions set at 20-28 °C day and night. Three plants per pot were arranged in the greenhouse in three replicates per treatment, using a RCB design. Plants were irrigated three times a day with irrigation water containing NPK fertiliser [3: 1: 3 (38)] (50%) together with calcium nitrate (50%) and trace elements. The plants were allowed to grow for 30 days before one plant per treatment from each crop cultivar was harvested and sampled (stems) to confirm their endophytic colonisation by B. bassiana isolates. The stems were separately surface sterilised by immersing them in 3% sodium hypochlorite for 3 min, followed by 70% ethanol for 2 min. They were separately rinsed three times with sterile distilled water and placed on a sterile paper towel in a laminar flow cabinet to air dry. After drying, six pieces of each treated stem were randomly selected and plated separately onto B. bassiana selective medium. 27 The inoculated plates were incubated for 15 days at 25 °C. The plates were monitored every 2-3 days for the emergence of fungal mycelia. After colonisation of the stems by B. bassiana isolates was confirmed, the remaining inoculated plants were harvested, washed with tap water, and the stems were then used for pathogenicity testing on the stem borer, S. calamistis.

Efficacy of B. bassiana isolates against S. calamistis
The endophyte positive stems produced as previously described were harvested and washed with distilled water, before being fed to the third instar larvae of S. calamistis. A total of 10 third instar larvae of S. calamistis were placed into plastic containers of 10 g of B. bassiana infected rice stems. The containers were placed in an incubator at 28 °C. Mortality of the larvae was recorded after 7, 14, 21 and 28 days. For the control, larvae were fed with non-inoculated rice stems. Dead larvae were collected at 7, 14, 21 and 28 days and were maintained in plastic containers on Whatman filter paper previously wetted with sterile distilled water. Two to three days after collection, the dead larvae were surface sterilised in 3% sodium hypochlorite for 1 min followed by 70% ethanol for 1 min. They were then rinsed three times with sterile distilled water for 15 s. The surface-sterilised dead larvae were placed on sterile paper towels under a laminar flow cabinet for air drying. The dried dead larvae were plated onto Petri dishes that contained a B. bassiana selective media 27 (Figure 2). The plates were kept in an incubator at 26-28 °C and monitored every 2-3 days. Fungi that appeared on the surface-sterilised larvae of S. calamistis were harvested and sub-cultured onto fresh PDA plates for pure culture and identification. After 15 days, the colonies were compared to the endophytic B. bassiana isolates that were initially inoculated onto the rice seed. The experiment was performed three times to confirm the pathogenicity of the B. bassiana isolates.

Data analysis
Colonisation of the rice plant tissues resulting from the various treatment combinations was analysed using SAS (version 9.4). A general linear model was used for the analysis of variance (ANOVA). If the ANOVA F-test was significant (p<0.05), then treatment means were separated using the Duncan Multiple Range Test.
The cumulative percentage of mortality of S. calamistis was recorded, and the area under the mortality progress curve (AUMPC) was calculated. The data collected were analysed using GenStat (18th edition). A twoway ANOVA was run with B. bassiana isolates and time (days) as the main factors.

Results
The rice plants were colonised by B. bassiana isolates using both inoculation methods. The colonisation of each rice plant tissue (root, stem and leaf) was both isolate and inoculation method dependent and varied also with time (30-60 days) (Tables 1 and 2).
Following seed treatment, highly significant differences were observed in the colonisation of the tissues (roots, stem and leaves) x B. bassiana isolates (p=0.0001) and x rice cultivar (p=0.0001). At both 30 and 60 days for all interactions [(isolates x cultivars, isolates x time, cultivars x time and isolates x cultivars x time] there were highly significant differences in the colonisation of the roots and the leaves (p=0.0001). There was no interaction between strain x time (p=0.32) ( Table 1).
After foliar sprays of inoculum, highly significant differences were observed in the levels of colonisation of the roots between B. bassiana isolates, rice cultivars, B. bassiana isolates x cultivars, B. bassiana isolates x time, cultivars x time, and B. bassiana isolates x cultivars x time (p=0.0001; Table 2). Highly significant differences were observed in colonisation of the leaves between B. bassiana isolates, cultivars, time (30- There were highly significant differences between the pathogenicity of the five B. bassiana isolates that were used against the third instar larvae of S. calamistis (p=0.001; Table 3). B. bassiana isolates Bb4 and Bb35 were the most effective strains, killing 93.3% and 76.6% of S. calamistis larvae at 28 days, respectively (Figure 3). The AUMPC data revealed that the B. bassiana isolates Bb4 vs Bb35, Bb35 vs Bb10 and Bb10 vs Bb3 showed similar levels of pathogenicity (Table 3).    36 Both inoculation methods (seed treatment and foliar spray) resulted in high levels of leaf and root colonisation. The inoculation method did not appear to favour a specific pattern of local colonisation of the rice cultivars. This is contrary to the results of Posada et al. 21 who reported that foliar sprays favoured leaf colonisation, whereas soil drenching favoured root colonisation in coffee. Similar findings were demonstrated for the common bean. 25 The systemic spread of each B. bassiana isolate differed over the two sampling time periods (30 and 60 days) used in this study. A reduction in level of colonisation over time may have been caused by a host resistance response to the heterotrophic fungi or because of competition from other endophytes in the rice tissues. 23 The colonisation of the rice cultivars by the B. bassiana isolates did not cause any apparent negative effects on the growth of the rice plants, as was reported by Van Bael et al. 37

Conclusion
This study revealed that rice cultivars could be colonised by strains of B. bassiana. The five B. bassiana isolates tested in this study were endophytic with various degrees of colonisation and pathogenicity against the rice stem borer, S. calamistis. The results of this study indicate that two of the five tested B. bassiana isolates hold promise as biological control agents of rice stem borers. Further studies under field conditions at different sites and seasons are needed to ascertain the potential of these isolates. From this study, seed treatment seems to be the most appropriate and practical way to introduce the best B. bassiana strains during field studies. The field experiments will be implemented where rice is grown on a large scale using an experimental formulation of the best two B. bassiana strains.