First report of biocontrol agent

First report of biocontrol agent, Cladosporium cladosporioides isolated from insect in Bangladesh

Md. Touhidul Islam1,2, Depali Rani Gupta1, Musrat Zahan Surovy1, Nur Uddin Mahmud1 and Md. Tofazzal Islam1,3*
1Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur-1706, Bangladesh
2School of Agriculture and Rural Development, Bangladesh Open University, Gazipur- 1705, Bangladesh
3Davis College of Agriculture, Natural Resources and Design, 333 Evansdale Drive, West Virginia University, Morgantown, WV 26506, USA

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This is the first report of biocontrol agent, Cladosporium cladosporioides that was isolated from brown planthopper of rice in Bangladesh. The isolate BOU1 was identified by the observation of color, shape, size, and growth rate of the colony as well as by making a culture slide which included the observation of the structure of asexual spores, spore shape, and form hyphae. The BLAST search of the ITS1 region revealed that match to a sequence registered under the accession number for C. cladosporioides. The submitted sequence data was registered and provided the accession number of MG654669 by GenBank. The fungal isolate was cultured on four different media viz. PDA, PDAY, SDA and SNA to observe the virulence as well to find out the best media for this isolate. The result revealed that PDA is the best media for C. cladosporioides. The fungal isolate was used for plant growth by germination percentage, germination index, vigor index- I and vigor index- II against rice and wheat seed. From this experiment, the result displayed that C. cladosporioides enhanced the germination and vigor index of those seeds. The fungal isolate was also used against whitefly as direct contact toxicity on eggplant leaves. Dipping method was used in this experiment under laboratory condition. The current study investigated that the highest mortality (70.83%) of B. tabaci was observed for 108 conidia/ml of C. cladosporioides. This paper is concluded that C. cladosporioides is a very good candidate as biocontrol agent against whitefly and for plant growth.
Key words: biocontrol agent, germination, vegetative growth, sporulation, compatibility, IPM

There are many kinds of fungi in dwelling environments. One of the predominant species is Cladosporium, and it can be isolated from plant (Torres et al., 2017; Saranya et al., 2013; Bensch et al., 2010) and insect (Habashy et al., 2016; Abdel-Baky, 2000). It is an important agent of plant disease, attacking both the leaves and fruits of many plants. This species produces asexual spores in delicate, branched chains that break apart readily and drift in the air. It is able to grow under low water conditions and at very low temperatures. The genus Cladosporium, which comprises more than 772 names (Dugan et al., 2004), has been studied extensively in recent years.
The genus Cladosporium is one of the largest genera of dematiaceous hyphomycetes, and is characterized by a coronate scar structure, conidia in acropetal chains and Davidiella teleomorphs. Morphological identification of any fungus especially Cladosporium spp. is a difficult subject. The size and shape of conidia are important characters to identify the fungal species, but the dimensions of conidia can vary among the species in the genus. Based on morphological examinations (Schubert and Braun, 2004; Heuchert et al., 2005; Schubert, 2005; Schubert and Braun, 2005a, b, 2007; Braun et al., 2006, 2008a, b; Crous et al., 2006a, b; Schubert et al., 2006; Braun and Schubert, 2007) and molecular studies (Crous et al., 2006a, 2007a,b,c; Arzanlou et al., 2007; Schubert et al., 2007a, b), a modern generic concept of Cladosporium was established, including clear delimitations from morphologically similar genera (Crous et al., 2007b; de Hoog et al., 2007; Seifert et al., 2007). The fungal isolate of C. cladosporioides complex was made based on molecular phylogeny using ITS, ACT and EF-1? regions (Bensch et al., 2010).
The virulence of C. cladosporioides involves four steps: adhesion, germination, differentiation and penetration. Each step is influenced by a range of integrated intrinsic and external factors, which ultimately determine the pathogenicity. The most important enzymes secreted by entomopathogenic fungi like Cladosporium spp. are lipases and proteases, which are produced sequentially (Smith et al., 1981). Regarding the role of Cladosporium spp. against aphids, there is number of reports emphasized the ability of such fungus in reducing the population size of these pests (Lagowska, 1995; Vallejo et al., 1996; Han et al., 1997). In Egypt, Abdel-Baky et al. (1998) recorded three species of Cladosporium (C. uredinicola, C. cladosporioides and C. chlorocephalum) which infect Bemisia spp., Aphis gossypii and Empoasca sp. In china, C. cladosporioides caused 20-57% natural mortality of Hemiberlesia pitysophila under field conditions and 39% in the laboratory tests (Pan et al., 1989). Whereas, Thumar and Kapadia (1994) mentioned that Cladosporium spp. was able to infect Aleurolobus barodensis nymphs at all the year times in India. The biocontrol fungi, C. cladosporioides CL-1 significantly improved the growth of tobacco seedlings in vitro when they were co-cultivated without physical contact (Paul and Park, 2013).
The present study report regarding the isolation and identification of a biological control agent of C. cladosporioides that was collected from brown plant hopper of rice in Bangladesh based on conventional and molecular characterizations and determinations of its potential usefulness as a biocontrol agent.
Materials and methods
Biocontrol agent
Cladosporium cladosporioides (isolate BOU1) was obtained from the farmer’s rice field in Gazipur, Bangladesh, which was originally isolated from the brown planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) of rice. The fungus was cultured on PDA medium.
The sweetpotato whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) was used in this experiment. It was identified as the BW3 biotype based on the mitochondrial cytochrome oxidase I (mt COI) gene (Jahan et al., 2015). The whitefly population was maintained on eggplant, Solanum melongena L (Table 1) for at least two successive generations before being used in experiment.
Experiment- 1: Isolation and identification of Cladosporium cladosporioides
Isolation of Cladosporium cladosporioides
The entomopathogenic fungus, C. cladosporioides was obtained by using insect bait technique (Zimmermann, 1986). Sterile needle was used to isolate mycelia or conidia from the cuticles of cadavers of the brown plant hopper and transferred into a 1.5 ml Eppendorf tube containing sterile distilled water. Conidial suspension (5 × 105 conidia/ml) was prepared and 100 ?l transferred homogeneously spread with drigalski to a Petri dish containing 15 ml of PDA and incubated at 28 °C. After three days, the dishes were checked daily until the eighth day. Fungal colonies appearing on each Petri dish were transferred to Eppendorf test tubes containing PDA and incubated at the same conditions as earlier stated. Each isolate was isolated and observed with a light microscope. Single conidia on the surface of water-agar medium was collected with a sterile capillary tube and transferred onto PDA to isolate the culture. Fungal isolate was purified by taking isolate fungus growing on a Petri dish that already contains other PDA medium. Purified was back several times with the same technique in order to obtain pure cultures fungal isolate. After getting fungal isolate pure, pure culture was then identified.
Identification of Cladosporium cladosporioides
Identification of fungal isolate was divided into two stages. The first phase of macroscopic observations conducted on pure cultures PDA a sterile Petri dish, which includes observation of color, shape, size, and growth rate of the colony. The second stage of microscopic observation was made by making a culture slide (Malloch, 1997) which included the observation of the structure of asexual spores, spore shape, and form hyphae (Malloch, 1997; Onions et al., 1981).
Experiment- 2: Molecular identification of Cladosporium cladosporioides
Genomic DNA extraction
Genomic DNA was extracted from isolate of C. cladosporioides following modified CTAB methods (Zhang et al., 2010). The isolates was grown on filter paper on potato dextrose agar medium for 10 days at 25º C and the mycelia were harvested from the filter paper by scrapping. The harvested mycelia was then grind in a mortar pastel adding 600µl of extraction buffer (33mM CTAB; 0.1 M Tris-HCl, pH 8.0; 7.8mM EDTA; 0.7M NaCl). The mycelial suspension was collected in an Eppendorf tube and incubated at 65º C for 30 minutes in a heat block with occasional shaking. Purification with phenol: chloroform: isoamyl alcohol (25: 24: 1) and DNA precipitation with 700 µl of cold isopropanol were conducted. DNA was washed with 70 % ethanol, dried under vacuum and re-suspended in TE buffer (10 mM Tris-HCl, pH 8.0; 1.0 mM EDTA), containing 10 µg/ml of RNase A and incubated at 37 ºC for 30 minutes and stored at -20 ºC. Quantification of DNA was performed on 0.5 % agarose gel and diluted with sterile distilled water.
PCR amplification of ribosomal RNA genes
A nuclear rDNA region was amplified by PCR according to White et al. (1990) using primers ITS1 (5′ TCC GTA GGT GAA CCT TGC GG 3′) and ITS4 (5′ TCC TCC GCT TAT TGA TAT GC 3′). PCR was performed in a 50-?l reaction mixture with DNA Taq polymerase (Promega, Madison, WI) and purified genomic DNA from fungal isolates. PCR was performed for all symptomatic plant samples in a 20-?l reaction mixture with the Extract-N-Amp kit in a DNA thermal cycler. The PCR contained the following reaction mixture of 0.5 ?l of DNA Taq polymerase (2.5 U), 5 ?l of 10× polymerase buffer, 3 ?l of 25 mM MgCl2, 1 ?l of 10 mM dNTP, 2 ?l of 20 pmol/?l of each primer, and 1 ?l of the template (extracted genomic DNA at 50 ng/?l) in a total volume of 50 ?l. The reaction mixtures used with the Extract-N-Amp kit consisted of 4 ?l of the diluted sample extract, 10 ?l of 2× PCR mix (kit), and 1 ?l of 20 pmol/?l of each primer in a total volume of 20 ?l. The PCR amplification reaction was carried out in a thermal cycler with the following temperature conditions as described by Harmon et al. (2003) with an initial de-naturation of 2 min at 94°C followed by 30 cycles of 45 s of denaturation at 94°C, 45 s of annealing at 55°C, and 45 s of extension at 72°C. Final extension was 10 min at 72°C. The amplification products were subjected to electrophoresis in a 1% agarose gel and stained for 10 min in an ethidium bromide solution (10 ?g/ml) and visualized under UV light.
Phylogenetic analysis of ITS
Sequences corresponding to both regions were assembled and edited using Bioedit soft-ware version 7.1.3 (Hall, 1999) and a consensus sequence of each isolate was created and submitted to BLASTN 2.2.19 (Zhang et al., 2000). For evolutionary analyses, all consensus sequences were compiled into a single file (FASTA format) and aligned us¬ing the profile mode, Clustal W 1.81 (Thompson et al., 1994) included in the MEGA 5 software (Tamura et al., 2011). ITS1 and ITS4 regions of the isolate was sequenced and subsequently submitted to GenBank.
Experiment- 3: Effect of different media on virulence of Cladosporium cladosporioides
The effect of different media viz. PDA, PDAY, SDA and SNA was evaluated against virulence of C. cladosporioides. The virulence study of the fungus was conducted by two ways- growth (germination, colony area and conidiogenesis) and enzymatic activities (protease and lipase) of the fungus.
Germination assay
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 media. The Petri dishes were incubated at 25 ± 1 ºC, 70 ± 10% r.h., and L12: D12 photoperiod for 24 h. After 24 h, three separate fields were observed for germination at 40× magnification for each treatment and 100 conidia were observed randomly in each field. There were 6 Petri dishes for each field. Conidia with germ tubes equal to or greater than the width were considered to have germinated.
Colony area and conidiogenesis
Conidia of C. cladosporioides were suspended in the respective aqueous solution (107 conidia/ml) for vegetative growth and conidiogenesis containing 10 ml media in to each Petri dish. The isolate was point inoculated into the centre of the agar with a 5-µl conidial suspension and the Petri dishes were incubated as same as germination assay. There were 6 Petri dishes for each treatment. The measurements of colony diameter were made daily after inoculation until 14 days. The average diameter of every colony was calculated as (long diameter + short diameter)/2. Spore production was investigated after 14 d of inoculation. Conidia from six randomly selected plates from each treatment were dislodged by two washes of 50 ml and agitation for 15 min for conidiogenesis. Then, the conidia were counted using a compound microscope with a hemocytometer.
Methods of measurement were compared to determine the differences in analyzed results and whether these differences changed the final conclusion made at the treatment level. In order to compare measured area (MA) to diameter (D), average diameter was converted to area of a circle,
D_A=??r?^2=?(AverageD/2)^2——————————————————————– (i)
Where, DA = Calculated area and MA = Measured colony area.
Enzyme assay
The two basic enzymes- protease and lipase of C. cladosporioides were observed as a virulence of entomopathogen. For all enzyme assays, 150 ml Erlenmeyer flasks contained 50 ml of media were sterilized by autoclave. After cooling, 5 ml of a fungus suspension (107 conidia/ml) were inoculated to each flask and incubated at 30 °C with shaking at 180 rpm for 5 days. After 5 days, the individual enzyme determination was described as follows-
Protease activity
Proteolytic or caseinolytic activity of C. cladosporioides was determined as shown by Söderhäl and Unestam (1975). After incubation period, the content of each flask was filtered through Whatman no. 2 filter paper. There were 10 Petri dishes for each treatment. The test tubes containing 1 ml of the filtrate culture were centrifuged at 12,000g for 20 min (4 ºC) and were incubated in a water bath at 30 °C. After 5 min, 1 ml of a 2% (w/v) vitamin-free casein solution in 0.5 M Tris-HCl buffer, pH 8.3, of the same temperature was then added to each tube. The mixture was incubated at 30 °C for 15 min without shaking. The reaction was stopped by the addition of 5 ml of a 10% (w/v) trichloroacetic acid (TCA) and samples were allowed to stand at 25 °C for 1 h. After centrifugation at 3,000 g for 15 min, the supernatant was filtered and its absorbance was determined at 280 nm. Controls in which TCA was added to the filtrate before adding the substrate were prepared in parallel for all treatments. A mixture containing 1 ml buffer was used as blank and treated in the same manner. 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 ºC (Tremacoldi and Carmon, 2005).
Lipase activity
Lipase activity of C. cladosporioides was determined as described by Cordenons et al., 1996. After incubation period, the substrate emulsion was prepared as a 1:1 mixture of olive oil (50 ml) and gum arabic (50 ml, 10% wt/vol). The test tube contained 1 ml of the spore suspension (107 conidia/ml), 5 ml substrate emulsion, and 2 ml of 50 mM phosphate buffer (pH 6.8) and was incubated for 1 h at 37 °C with shaking. The reaction was stopped with 4 ml of acetone-ethanol (1:1) containing 0.09% phenolphthalein as an indicator. The contents were titrated with 0.05 N NaOH using a burette until a light blue color appears. 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:
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 bydetermining the amount of supernatant that produces 1 mol of fatty acid per minute under the specified assay conditions.
Experiment- 4: Enhancement of rice, wheat and eggplant seed germination and seedling vigor by Cladosporium cladosporioides
Seed preparation
The seeds according to Table 1 were surface sterilized with 70% ethanol, followed by 5% sodium hypochlorite and washed by sterilized distilled water. One hundred seed grains were selected for each treatment then soaked in the respective C. cladosporioides in a flask containing 107 spores/ml spore suspension for 30 min. The seeds soaked in sterilized distilled water served as control. The treated seeds were incubated for 15 days in sterilized petri dishes at 25 ± 2 °C fitted with filter paper and each petri dish was irrigated with 10 ml sterilized water.
Seedling length and weight
The root and shoot length were measured by selecting five seeds randomly for each treatment. Shoot length was measured from the base of the primary leaf to the base of the hypocotyls and root length was measured from the tip of the primary root to the base of hypocotyl, with all measurements expressed in centimeters. Shoot and root weight were examined using digital weight scales and expressed in grams. All measurements were according to the method used in Dash (2012).
Seed germination rate
Germination rate is the average of number of seeds that germinate over the 7 and 15 day periods.). Germination rate was computed using the formula proposed by IRRI (2011):
Germination Percentage= (Number of germinated seed )/(Number of non-germinated seed)×100 ——————— (iii)
Germination Index (GI)
Numbers of seedlings emerging daily are counted from day 0 until day 5 of planting. Thereafter a Germination Index (G.I) is calculated by using the following formula as proposed by AOSA (1983):
GI= (Number of Germinated Seed)/(Days of First count)+?+(Number of Germinated Seed)/(Days of Final count)———————— (iv)
Seedling vigor index
Seed vigor index was examined after five days of incubation using the formula given by Abul-Baki and Anderson (1973) based on the product of germination (%) and seedling length (cm):
Seedling vigor index-I = (Seedling length × Germination Percentage )/100—————– (v)
Seedling vigor index-II = (Seedling dry wt. × Germination Percentage )/100—————- (vi)
Experiment- 5: Pathogenicity of whitefly, Bemisia tabaci using Cladosporium cladosporioides
The mortality percentage of B. tabaci by C. cladosporioides was estimated on eggplant. When plants were 6 weeks old (4-5 true leaves), approximately 50 adult whiteflies (2 days old) were released onto each plant for 48 h to allow egg laying, after which adults were removed. The plants were then incubated for a further 12 days to allow eggs to hatch and reach the second larval instars. Afterwards, infested leaves were labeled and dipped into 108 conidia/ml of C. cladosporioides on both leaf surfaces for 10 s. There were 10 leaves for each treatment. Numbers of dead and living nymph were recorded at day 8 post infection (25 ± 1 ºC, 70 ± 10% RH and L 12: D 12 photoperiod) after dipping treatments on second instars were performed. Tween 80, 0.02% without fungal suspension served as untreated control. Immature were considered dead if they had lost their normal yellow-green color, turgidity, and smooth cuticle structure.
Statistical analysis
Data were analyzed with a one-way ANOVA. The statistical analyses were performed using the Proc GLM procedure (SAS Studio 3.6, 2017). Means were separated using Least Significant Difference (LSD) test at 5% level of significance.
Experiment- 1: Isolation and identification of Cladosporium cladosporioides
Based on the observation of colony morphology as well as the size and shape of conidia, the isolated fungus was initially identified as C. cladosporioides (Fig. 1). Conidial chains were very long, with up to 18 conidia. The cladosporioid scar structure with dome and rim were not clearly visible under light microscopy (Fig. 1). Colonies on PDA grey-olivaceous to dull green or olivaceous-grey, reverse iron-grey, leaden-grey or olivaceous-black, velvety to floccose, margins grey-livaceous to white, feathery, regular, aerial mycelium sparse, diffuse, or sometimes abundantly formed, dense, floccose-felty, low, forming mats, growth flat to low convex, usually without prominent exudates, occasionally with several small prominent exudates.
Experiment- 2: Molecular identification of Cladosporium cladosporioides
The development of PCR for the amplification of the various rDNA regions has considerably facilitated taxonomic studies of fungi. The alignments and phylogenetic analysis confirmed the taxonomic identity of the strains used in our study, with C. cladosporioides. The amplification of the ITS region resulted in a single product for the isolate. The size of the product was 480 bp. Microscopic examination of the unidentified fungal isolate resulted in preliminary identification as Cladosporium sp. The BLAST search of the ITS1 region revealed the match to a sequence registered under the accession number for C. cladosporioides. The submitted sequence data was registered and provided with the accession number of MG654669, which was released in December 2017 (GenBank). The unidentified isolate was very similar with C. cladosporioides, an average pair wise similarity of 100%. The data allowed us to conclude that BOU1 is C. cladosporioides isolate.
Experiment- 3: Effect of different media on virulence of Cladosporium cladosporioides
The four different media was used to investigate the virulence as well as the suitability of the media for C. cladosporioides based on colony area, conidiogenesis, protease and lipase activity (Table 2). Significant differences were observed on all variables viz. colony area (F = 66.35; P ; 0.0001; df = 3, 23; Table 2), conidiogenesis (F = 256.54; P ; 0.0001; df = 3, 23; Table 4), protease (F = 21.51; P ; 0.0001; df = 3, 23; Table 2) and lipase (F = 37.49; P ; 0.0001; df = 3, 23; Table 2) activity of C. cladosporioides as compared with their respective control. The colony area (Fig. 2), conidiogenesis, protease and lipase were highest for PDA, reaching up to 33.52 cm2, 9.91 × 105 conidia/ml, 12.77 µg/ml/hr and 9.75 µmol fatty acid/ml, respectively (Table 2). These values decreased up to 22.63, 0.17 × 105 conidia/ml, 10.94 µg/ml/hr and 4.08 µmol fatty acid/ml, respectively for SNA (Table 2). The research revealed that PDA is the most suitable media among all media as used in this experiment for C. cladosporioides.
Experiment- 4: Enhancement of rice and wheat seed germination and vigor by Cladosporium cladosporioides
All the variables of germination and seedling vigor of rice and wheat seed were increased by the conidial suspension of C. cladosporioides isolate (Table 3). Significant differences were observed among the treatment of germination percentage (F = 4.65; P = 0.0126; df = 3, 23), germination index (F = 2.98; P = 0.05; df = 3, 23), vigor index- I (P ; 0.0001; F = 14.51; df = 3, 23) and vigor index- II (F = 55.29; P ; 0.0001; df = 3, 23) as compared with their respective controls (Table 3). The research revealed that the rice seed treated by C. cladosporioides isolate showed the highest germination index and vigor index- I with average mean values of 8.15 and 4.50, respectively; while the treated wheat seed displayed the highest germination percentage and vigor index- II with average mean values of 80.33 and 0.046 (Table 3).
Experiment- 5: Pathogenicity of whitefly, Bemisia tabaci using Cladosporium cladosporioides
One-way ANOVA results of nymph mortality of B. tabaci showed significant difference among the concentrations of C. cladosporioides (F = 1154.89; P ; 0.0001; df = 3, 23) as compared with control (Table 4). The current study investigated that the highest mortality (70.83%) of B. tabaci was observed for 108 conidia/ml of C. cladosporioides (Table 4). The lowest mortality (0%) of B. tabaci was observed for control (Table 4). There was a positive correlation between the factors i e, concentration of C. cladosporioides*mortality percentage of B. tabaci. The mortality percentage of B. tabaci was increased with increasing the concentration of C. cladosporioides up to 1 × 108 conidia/ml, but the concentration over 1 × 108 conidia/ml, the mortality percentage was decreased (Tabulated data was not shown). It indicates that 1 × 108 conidia/ml concentration of C. cladosporioides, strain BOU1 is the best concentration for whitefly control.
Conidia color may differ in colony size and condition (Latch, 1964). After 10 days incubation of C. cladosporioides, the culture produces a white mycelial margin with clumps of more or less verticillate branching conidiophores. These branching conidiophores became colored with the devel¬opment of the spores. According to the Fig. 1, the color of C. cladosporioides varies from dark green to grey. A similar phenomenon was reported that the colony of C. cladosporioides was as yellowish green or olivaceous green, sometimes even as pink buff colonies (Bridge et al., 1993). The morphological characters of this fungus were described earlier (Brady, 1979; Humber, 1997; Hoog et al., 2000). Mycelium often wholly coveting affected hosts; conidiophores in compact patches; densely intertwined; conidiogenous cells with rounded to conical apices, arranged in dense hymenium; conidia aseptate, cylindrical or ovoid, forming chains usually aggregated into prismatic or cylindrical columns or a solid mass of parallel chains (Liang et al., 1991).
The molecular analysis is a useful approach for identification of Cladosporium species. The fungal isolate of C. cladosporioides complex was made based on molecular phylogeny using ITS, ACT and EF-1? regions (Bensh et al., 2010). Our results are consistent with Bensh et al. (2010), and support the possible presence of cryptic species complexes on C. cladosporioides lineages. A similar strategy for different species of entomopathogenic fungus, Paecilomyces fumosoroseus (=Isaria fumosoroseus) (Azevedo et al., 2000), Metarhizium anisopliae (Destéfano et al., 2004; Muerrle et al., 2006) and C. cladosporioides (Torres et al., 2017) was also reported earlier. The development of PCR for the amplification of the various rDNA regions has considerably facilitated taxonomic studies of fungi. The alignments and phylogenetic analysis confirmed the taxonomic identity of the strains used in our study, with C. cladosporioides. The first attempts were made to sequence the ITS region using general fungal primer sets for entomopathogenic fungi (Driver et al., 2000; de Muro et al., 2003; Glare, 2004).
The virulence of entomopathogenic fungi is influenced by the substrates as well as media composition, with complex relationships between various spore parameters, e.g. stress response, germination rate (Kim et al., 2014; Maldonado-Blanco et al., 2014; Pelizza et al., 2011). The cuticle degrading enzyme activities of the entomopathogenic fungi is also influenced by the substrate (Mascarin et al., 2013; Rosas-Garcia et al., 2014; Ortiz-Urquiza et al., 2016). Germination rate is an important virulence-determinant factor that can be affected by nutritional conditions (Fragues et al., 2001). The germination rate of C. cladosporioides was highest on PDA medium during present investigation. An inverse phenomenon was observed for M. anisopliae by Ali et al. (2009). The germination speed of conidia of M. anisopliae produced on different media varied significantly (Shah et al., 2005). The highest germination rate of M. anisopliae was noted for conidia produced on 1% yeast extract as well as on osmotic stress medium (Ali et al., 2009). Following the germination rate, the highest colony area of C. cladosporioides was also noted for conidia produced on PDA in our investigation. The radial growth of fungal strains changes not only with fungal species and isolates but also with the media used for production of conidia (Shah et al., 2005). The isolates of I. fumosoroseus showed maximum colony growth rate for high C/N medium, whereas the lowest radial growth was observed on osmotic stress medium (Ali et al., 2009). Similar phenomenon was observed for B. bassiana and M. anisopliae (Safavi et al., 2007). The conidia of B. bassiana WT isolated from PDA were more efficacious than those derived from either CZA or SDAY (Ortiz-Urquiza et al., 2016).
The presence of proteins and lipids on the insect cuticle requires a sequential action of appropriate hydrolytic enzymes (Samuels and Paterson, 1995). These enzymes could facilitate the early stages of fungal infection. During this study, protease and lipase activity of C. cladosporioides was observed as a function of culture conditions and this revealed some interesting results. The highest protease and lipase activity of C. cladosporioides was observed on PDA medium. Ali et al. (2009) reported that protease (Pr1) activity of M. anisopliae was the highest in low and complex C/N ratio media; while the highest lipase activity of M. anisopliae was observed on 2% peptone and osmotic stress media. Cuticle-degrading Pr1 enzyme and lipases have been shown to be a pathogenicity determinant in most entomopathogenous hyphomycetes, with an established role in virulence towards insect hosts. This type of enzymatic activity can be the repression of carbon and nitrogen sources that were described for M. anisopliae previously (St. Leger et al., 1988; Paterson et al., 1994a, b; Screen et al., 1997, 1998).
The biocontrol agent, C. cladosporioides has been applied to the agricultural crops like rice and wheat for the purpose of growth enhancement, including increased seed germination, and seedling vigor. Our results showed that the fungal plant growth promoting agent significantly improved the germination and vigor of rice and wheat seedlings. A similar phenomenon was reported for different type of microorganism that the plant growth-promoting rhizobacteria (PGPR) for the purpose of growth enhancement of agricultural crops, including increased seed germination, plant weight, and yield (Kloepper et al., 1991, 1999). The plant-growth-promoting fungus (PGPF), Talaromyces sp. significantly enhanced the growth of Brassica campestris seedlings and their resistance to Colletotrichum higginsianum (Yamagiwa et al., 2011). The inoculation of the rice seeds with Trichoderma spp. significantly increased rice seed germination rate, vigor index and speed of germination as compared with untreated control (Doni et al., 2014).
The appropriate use of environment-friendly microbial pesticides can play a significant role in sustainable crop production by providing a stable pest management program. Biocontrol agent represents a potentially important control measure of pest populations. According to IPM strategy, the biocontrol agent of C. cladosporioides, strain BOU1 can reduce the reliance on synthetic pesticides and at least 70% negative impact can minimize on the environment for whitefly control. Strategy might be employed for the rest of 30% whitefly population by combining this biocontrol agent with sub lethal doses of chemical insecticides and botanicals those may act synergistically increasing the efficiency of the control, allowing the lower doses of insecticides, preservation of natural enemies, minimizing environmental pollution and decreasing the likelihood of development of resistance to either agent (Ambethgar, 2009). This can improve workers safety while at the same time maintaining the economic viability for crop production. However, as maximum mortality value caused by C. cladosporioides was 72.5% after 7 days that is similar with our findings (Habashy et al., 2016). The isolate of C. cladosporioides caused the mortality of Tetranychus urticae was as high as 70% within 2.77 days (Eken and Hayat, 2009). The biocontrol agent of C. cladosporioides caused high infection rate of whitefly, which reached 44.38% and 42.24% on castor oil in 1998 and 1999, respectively (Abdel-Baky, 2000).
This study is part of the postdoctoral research of Dr. Md. Touhidul Islam. He is grateful to University Grant Commission (UGC) of Bangladesh for providing a Postdoctoral Research Fellowship. Financial support by the World Bank funded project no. HEQEP CP # 2071 is also gratefully acknowledged.
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Table 1 List of plant species with their scientific name, family, variety and source as used in this research
Name of Seed Scientific Name Family Variety Source
Eggplant Oryza sativa
Triticum aestivum
Solanum melongena Gramineae
BRRI Dhan 28
BARI Ghom 30
Baromashi Singnath Begun BADC1
1Bangladesh Agricultural Development Corporation
2Bangladesh Agricultural Research Institute
3Sobuj Beej Bhandar, Bangladesh

Table 2 Growth and enzymatic activity of C. cladosporioides against different media as compared with control
Treatment Growth of C. cladosporioides Enzymatic activity of C. cladosporioides
Colony area (cm2) Conidiogenesis
(1 × 105 conidia/ml) Protease activity Lipase activity
SNA 33.52 ± 0.43 a
32.67 ± 0.43 a
25.56 ± 1.01 b
22.63 ± 0.56 c 9.91 ± 0.31 a
4.17 ± 0.33 b
2.00 ± 0.26 c
0.17 ± 0.11 d 12.77 ± 0.09 a
11.53 ± 0.11 b
11.06 ± 0.21 bc
10.94 ± 0.10 c 9.75 ± 0.21 a
6.67 ± 0.44 b
5.08 ± 0.31 c
4.08 ± 0.57 c
Means with the same letter within the same concentration are not significantly different (LSD-test, following one-way ANOVA: P < 0.05).

Table 3 Effect of seed treatment by C. cladosporioides on germination and seedling vigor of rice and wheat as compared with their respective controls
Treatment Seed Germination Vigor
Germination percentage Germination index Vigor index- I Vigor index- II
C. cladosporioides Rice 75.33 ± 1.33 c
78.33 ± 1.17 ab 7.47 ± 0.05b
8.15 ± 0.20 a 3.83 ± 0.22 b
4.50 ± 0.04 a 0.032 ± 0.001 c
0.041 ± 0.001 b
C. cladosporioides Wheat 75.50 ± 0.96 bc
80.33 ± 1.12 c 7.63 ± 0.20 ab
7.98 ± 0.21 ab 3.30 ± 0.11 c
4.29 ± 0.13 a 0.024 ± 0.001 d
0.046 ± 0.001 a
Means with the same letter within the same column are not significantly different (LSD-test, following one-way ANOVA: P < 0.05).

Table 4 Mortality of whitefly against C. cladosporioides as compared with control
Treatment n Mortality percentage
1 × 106
1 × 107
1 × 108 73
64 0.00 ± 0.00 d
19.50 ± 0.76 c
43.17 ± 1.14 b
70.83 ± 1.17 a
n = number of whitefly nymph. Means with the same letter within the same concentration are not significantly different (LSD-test, following one-way ANOVA: P < 0.05).

Fig. 1: Isolation history of C. cladosporioides, strain BOU1 by clock wise- Infected brown planthopper (BPH) with C. cladosporioides, isolated C. cladosporioides from BPH in PDA, spore of C. cladosporioides, one-day-old mycelium of C. cladosporioides in PDA and two-day-old mycelium of C. cladosporioides in water.

Fig. 2: Colony area of C. cladosporioides under different media by clock wise- PDA, PDAY, SNA and SDA

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