Abstract Cladosporium cladosporioides is one of the effective biological control agents against insects and mites. The objective of this research was to investigate the compatibility of C. cladosporioides, isolate BOU1 with two concentrations- 1% and 2% of the three plant extracts- Calotropis gigantean, Vitex negundo and Carissa macrocarpa by three ways- germination, vegetative growth and sporulation of fungal isolate. The compatibility of plant extracts with fungal isolate was calculated using the biological index formula. Thereafter, the compatibility of C. cladosporioides with these three plant extracts was evaluated in presence of grasshopper cuticle based on two basic enzymes- protease and lipase of C.
cladosporioides. Growth of C. cladosporioides was slightly affected by the three plant extracts. According to the compatibility index formula, all the three plant extracts with two concentrations- 1% and 2% were compatible with fungal isolate. Protease and lipase activity of C. cladosporioides was increased when the insect cuticle were added into the plant extracts. This paper is concluded that C. cladosporioides BOU1 was not sensitive to the three plant extracts used, especially in terms of fungal growth and cuticle degrading enzymes in presence of grasshopper cuticle.
Keywords: biocontrol agent, Cladosporium cladosporioides, fungal growth, insect cuticle, enzyme assay, IPMIntroduction The entomopathogenic fungus, Cladosporium cladosporioides is an important natural enemy of arthropods capable of infecting them directly through the integument (Shahid et al. 2012). Some Cladosporium species are efficient as biological insecticides, particularly against insects that have developed resistance to chemical insecticides (Abdel-Baky and Abdel-Salam 2003). It is well known that the entomopathogenic fungus is effective against a wide spectrum of insect pests and is commonly utilized in integrated pest management (IPM) as well as biological control programs (Bedini et al. 2018). By combining the entomopathogenic fungus with botanicals increase efficiency and accelerate the insect mortality (Asi et al. 2010; Serebrov et al. 2005; Purwar and Sachan 2006; Islam et al. 2010a, b). The appropriate use of entomopathogenic fungus and botanicals can play a significant role in sustainable crop production by providing a stable pest management program. The technology of IPM reduces the reliance on synthetic pesticides, minimizes the negative impact on the environment and improves workers safety while at the same time maintaining the economic viability of crop production (Tracy 2014). It operates throughout the world as a part of the management of pests in agriculture, forestry and greenhouse horticulture. With the increasing resistance of many insect pest species to chemical insecticides, pest control strategies are slowly shifting towards more sustainable, ecologically sound and economically viable options (Chidawanyika et al. 2012). To reduce the chemical insecticides for pest control and minimize the environmental hazard, biologically based pest management strategy is needed (Chidawanyika et al. 2012). To solve the pest problems permanently by the importation and successful establishment of natural enemies, the compatibility of biocontrol agent, C. cladosporioides with botanicals in presence of grasshopper cuticle has been undertaken. Materials and Methods Biocontrol agent The entomopathogenic fungus, Cladosporium cladosporioides (Davidiellaceae: Capnodiales) (Fresen.) de Vries, isolate BOU1 (GenBank accession number- MG654669) was obtained from the farmer’s rice field in Gazipur, Bangladesh, which was originally isolated from the brown planthopper Nilaparvata lugens (StҐl) (Hemiptera: Delphacidae). The fungus was cultured on PDA (potato dextrose agar) medium. Conidia of C. cladosporioides were collected from 15-day-old cultures (maintained at 25 ± 1 єC, 70 ± 10% r.h., and L12: D12 photoperiod) and were suspended in water. The conidia were quantified using a hemocytometer and a light microscope. Preparation of plant extract The leaf of three plants- (1) ‘Aakand’, crown flower (Calotropis gigantean, family Apocynaceae) (2) ‘Nishinda’, five leaved chaste (Vitex negundo, family Lamiaceae) and (3) ‘Koromcha’, natal plums (Carissa macrocarpa, family Apocynaceae) were obtained from the Botanical Garden of Bangladesh Agricultural University, Mymensingh, Bangladesh. The leaves were dried in the shade, and 50 g of each dried plant leaf was pulverized using blender. Then, 50 g leaf powder was dissolved in 500 mL ethyl acetate (EtOAc) and kept for overnight at room temperature. Ethyl acetate extracts were filtered and the filtrates were evaporated to dryness in vacuo using rotary evaporator at 45 ‚°C. EtOAc extract was dissolved again in 50 mL MeOH and partitioned with n-hexane to remove fat. After partitioning, the n-hexane fraction was discarded and MeOH fraction was kept in a beaker and left to dryness at room temperature. After drying, 113, 130 and 124 mg extracts were obtained from- C. gigantean, V. negundo and C. macrocarpa, respectively. These extracts were used for activity tests. Two concentrations- 1% and 2% of plant extracts were used in this experiment. Preparation of insect cuticle The grasshopper, Heteracris littoralis (Orthoptera: Acridoidea) was used in this experiment. The insects were obtained from the farmer’s rice field in Gazipur, Bangladesh. Comminuted cuticle from adult grasshopper was prepared essentially as described by Andersen (1980). Approximately 100 grasshoppers were frozen at -20 єC for 1 h and then homogenized in a Waring Blender in 1% (w/v) potassium tetraborate. The cuticle pieces were washed extensively in distilled water, stirred overnight in 1% (w/v) potassium tetraborate and then dried at 37 єC. The cuticle pieces were then milled to a fine powder in a Glen-Creston Hammer Mill (DEH-48) using a 0-5 mm sieves (Fig. 1). The powder was washed in 1% potassium tetraborate and finally in distilled water, allowed to settle and any floating material removed. Experimental procedure Compatibility of biocontrol agent with the three plant extracts was conducted by three ways- germination, vegetative growth and sporulation of C. cladosporioides. The germination speed of inoculum was assessed by inoculating with 0.5 ml conidial suspension (105 conidia/ml) in each of Petri dishes with 10 ml PDA. The six separate fields were observed for each treatment for germination at 40— magnification and 100 conidia were observed randomly in each field according to Islam et al. (2014, 2016). The measurements of colony diameter and spore production (conidiogenesis) were made after 14 days of incubation from the six Petri dishes (Islam et al. 2014, 2016). The in vitro compatibility of the plant extracts with fungal isolate was calculated using the biological index (BI) formula proposed by Rossi-Zalaf et al. (2008) as_BI=(47 — VG + 43 — SPO + 10 — GERM )/100 ——————————————————- (i)Where, BI = biological index;VG = percentage of vegetative growth compared to the control;SPO = percentage of sporulation compared to the control;GERM = percentage of conidia germination compared to the control.The effect of the plant extracts on the fungal isolate was classified based on the following classifications (Rossi-Zalaf et al. 2008): toxic (BI between 0 and 41), moderately toxic (BI between 42 and 66), and compatible (BI greater than 66). The two basic enzymes viz. protease and lipase of C. cladosporioides with the three plant extracts- C. gigantean, V. negundo and C. macrocarpa were also evaluated in presence of grasshopper cuticle. For both enzymes assay, 150 ml Erlenmeyer flasks contained 50 ml of PDA were sterilized by autoclave. There were 6 flasks for each treatment. After cooling, 5 ml of a fungus suspension (1 — 107 conidia/ml) were inoculated to each flask and incubated at 30 °C with shaking at 180 rpm for 5 days (Islam et al. 2016). Without insect cuticle, 1% insect cuticle and 2% insect cuticle were served as control when plant extract was used alone, plant extract combined with 1% insect cuticle and plant extract combined with 2% insect cuticle, respectively. After 5 days, the individual enzyme activity was determined. Proteolytic or caseinolytic activity of C. cladosporioides was determined according to S¶derh¤l and Unestam (1975) with some modification as described by Islam et al. (2016). One unit (U) of enzyme activity was defined as the amount of enzyme that, under the assay conditions described, gives rise to an increase of 0.1 units of absorbance in 1 h at 30 єC (Tremacoldi and Carmon 2005). Lipase activity of C. cladosporioides was determined as described by Pignede et al. (2000). Quantity of fatty acids liberated in samples was determined by equivalents of NaOH used to reach the titration end point, accounting for any contribution from the reagent, using the following equation (Cordenons et al. 1996): µmol fatty acid/ml subsample= ((ml NaOH for sample-ml NaOH for blank)—N—1000)/(5 ml)———– (ii) Where, N is the normality of the NaOH titrant used (0.05 in this case). Lipase activity (U/ml) was calculated by determining the amount of supernatant that produces 1 mol of fatty acid per minute under the specified assay conditions.Statistical analysisData were analyzed with a one-way ANOVA. The statistical analyses were performed using the Proc GLM procedure (SAS University Edition- SAS Studio 3.6, 2017). Means were separated using Least Significant Difference (LSD) test at 5% level of significance.Results Growth of C. cladosporioides was slightly affected by two suspensions of the three plant extracts (Fig. 1). One-way ANOVA results showed no significant difference among the three plant extracts with two concentration viz. 1% (F = 1.10; df = 3, 23; P = 0.3734) and 2% (F = 0.20; df = 3, 23; P = 0.8948) on germination percentage of C. cladosporioides as compared with their respective controls (Table 1). The mean percentages of germination for C. cladosporioides exposed to all plant extract in both concentrations were higher than 95% (Table 1). Incorporating the plant extracts into PDA led to no decrease in the mean colony area of the isolate after 14 days of incubation (Table 1). The result revealed that there is no significant difference among the three plant extracts with two concentration viz. 1% (F = 0.16; df = 3, 23; P = 0.9198) and 2% (F = 0.52; df = 3, 23; P = 0.6747) on vegetative growth of C. cladosporioides as compared with their respective controls (Table 1). The three plant extracts did not inhibit sporulation of C. cladosporioides significantly for both concentrations- 1% (F = 0.76; df = 3, 23; P = 0.5291) and 2% (F = 1.37; df = 3, 23; P = 0.281). According to the compatibility index formula, all the three plants extract with two concentrations- 1% and 2% were compatible with C. cladosporioides isolate (Table 2). There was a significant difference observed among the three plant extracts in absence of grasshopper cuticle on protease activity (F = 466.94; df = 3, 23; P < 0.0001) and lipase activity (F = 7.44; df = 3, 23; P < 0.0016) of C. cladosporioides as compared with their respective controls (without insect cuticle) (Table 3). When the insect cuticle were added into plant extracts, the in vivo virulence as well as protease and lipase activities of fungal isolate were increased (Table 3). According to the Table 3, protease activity of C. cladosporioides was significantly different among the three plant extracts in presence of 1% (F = 3495.76; df = 3, 23; P < 0.0001) and 2% (F = 4231.01; df = 3, 23; P < 0.0001) grasshopper cuticle as compared with their respective controls (individual treatment of 1% and 2% cuticle, respectively). Similar phenomenon was observed on lipase activity of C. cladosporioides for 1% (F = 2.63; df = 3, 23; P < 0.0784) and 2% (F = 8.57; df = 3, 23; P < 0.0007) grasshopper cuticle (Table 4). The plant extract of C. macrocarpa with 2% grasshopper cuticle showed the highest protease activity with average mean value of 18.88 µg/ml/hr, while the fungal isolate alone showed the lowest protease activity with average mean value of 9.13 µg/ml/hr (Table 3). The lipase activity was highest when the plant extract of V. negundo combined with 2% grasshopper cuticle with average mean value of 17.85 µmol fatty acid/ml; and the lipase activity was lowest when the fungal isolate in absence of grasshopper cuticle, but when it combined with the plant extract of C. macrocarpa, the average mean value of lipase activity was 11.17 µmol fatty acid/ml (Table 3). Discussion The agent of biological control is an essential component of integrated pest management (IPM) programs in multiple ecosystems that can provide more comprehensive management than either approach alone (Hoy 1994; Chidawanyika et al. 2012; Roubos et al. 2014). The compatibility of chemical pesticides or botanicals with biological control can be enhanced the different virulence parameters of biocontrol agent. This study is reported that the major three virulence parameters viz. germination percentage, colony diameter and conidiogenesis of C. cladosporioides were not affected by the three plant extracts. It indicates that C. cladosporioides is compatible with the three plant extracts used. A similar phenomenon was employed that the three entomopathogenic fungi- Beauveria bassiana (ESALQ-PL63 isolate), Isaria fumosorosea (ESALQ-1296 isolate) and Metarhizium anisopliae (ESALQ-E9 isolate) were compatible with Annona mucosa Jacq. (Magnoliales: Annonaceae) seed extract (Ribeiro et al. 2014). Higher concentration viz. 5% or greater than 5% a.i. (active ingredient) of commercial formulation of Azadirachta indica has a significant inhibiting effect on germination, vegetative growth and conidiogenesis of B. bassiana (Castiglioni et al. 2003). Conidial germination and hyphal growth of C. cladosporioides are temporally separated, physiologically different stages; therefore, the plant extracts as we used can affect these two events in a different way. Conidial germination of C. cladosporioides was not affected when they were exposed to the both of 1 & 2% concentrations of the three plant extracts in our study. A similar phenomenon was reported by Mohan et al. (2007) concerning B. bassiana with A. indica extract. In other hand, there was a strong positive correlation between germination speed and virulence in fungal pathogens in the several earlier studies (Rangel et al. 2008). The different earlier studies have also shown the difference in concentration of A. indica in seeds of different origins that the plant extracts have smaller negative impact on the entomopathogenic fungus (Sidhu et al. 2004; Devaranavadagi et al. 2003). Vegetative growth and conidiogenesis are also important parts of fungal life cycle for the development of secondary infections (Schumacher and Poehling 2012). In the present study, the vegetative growth was not significantly reduced by C. cladosporioides isolate in presence of botanicals in PDA medium. This finding is almost similar with Ribeiro et al. (2014) that B. bassiana was not affected by the different ESAM (ethanolic seed extract) concentrations. Information concerning the fungal stimulation by the growth promoting substances present in plants extract has also been published earlier (Akinbode and Ikotun 2008; Ribeiro et al. 2012). The infection process of entomopathogenic fungi on insects comprises in several stages. Among them, penetration is the most important process of cuticle infection of the insect which is measured by the enzymatic and/or physical mechanisms of entomopathogenic fungi (Ortiz-Urquiza and Keyhani 2013; Mondal et al. 2016). Many investigations and studies have been focused on the biochemical characterization of cuticle degrading enzymes such as protease and lipase of entomopathogenic fungi those involved in the process of pathogenesis (Cheong et al. 2016; Mondal et al. 2016). These enzymes degrade the insect’s integument that has already been carried out to understand the host-pathogen interaction (Mondal et al. 2016). During the present investigation, the activity of protease and lipase enzymes involved in the process of infection of C. cladosporioides isolate in presence of H. littoralis cuticle. When H. littoralis cuticle was added to the medium and botanicals, the protease and lipase activities of C. cladosporioides were increased. It assumes that these activities were stimulated by cuticle components. A dissimilar phenomenon was also employed for different entomopathogenic fungus that M. flavoviride CG423 (syn. M. anisopliae var. acridum) produced high levels of Pr2 only in minimal liquid medium without cuticle, with other negative effects was found in presence of Schistocerca pallens (Thunberg) cuticle (Tiago et al. 2002). Minimal medium with Diatraea saccharalis cuticle increased the protease and chitinase activities of B. bassiana than minimal medium alone (Svedese et al. 2013). The C. cladosporioides BOU1 was not sensitive to the three plant extracts used, especially in terms of fungal growth and cuticle degrading enzymes in presence of grasshopper cuticle. The strategy of combined application of entomopathogenic fungi with botanicals will open new perspectives in arthropod pest management that will facilitate the organic and ecologically based food production systems in developing countries. Finally, we can conclude that the three plant extracts as we used in this experiment are compatible with C. cladosporioides; and these two different types of the product might be useful for the control of insect. AcknowledgementsThis study is a part of the postdoctoral research of Dr. Md. Touhidul Islam. He is grateful to Bangladesh Open University for providing the study leave during this research. University Grant Commission (UGC) of Bangladesh provided financial support as a Postdoctoral Research Fellowship to the first author. World Bank funded project no. HEQEP CP # 2071 provided the partial financial support. Compliance with Ethical StandardsConflict of interestThe authors have declared that no conflict of interest exists. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.