Laboratory Evaluation of Temperature Effects on Germination, Radial Growth and Sporulation of Entomopathogenic Fungi and on Their Pathogenicity to Red Spider Mite, Tetranychus urticae Koch

Tomato, Lycopersicum esculentum (Miller) is a staple fruit vegetable in the world. Fresh fruits are very important source of vitamins and minerals which are essential for human health. It is considered as an important cash and industrial crop in many parts of the world (Babalola et al., 2010). India is the second largest producer of tomato next to China with an area of 8.80 million hectares, annual production of 18.22 million MT (11.5% of total production) and 19.5 MT of productivity (www.faostat.fao.org.). Tomato is ravaged by several insect species including mites. Red spider mite, Tetranychus urticae Koch is an important pest of tomato throughout the World and they are known to disperse through ballooning among healthy plants (Javed et al., 2019). The development of insecticide resistance in red spider mite populations and increasing public concern over ill effects of pesticide residues has led to investigations into alternative control measures (Tang et al., 2017). Thus, the development of effective biocontrol agents has received increasing interest as part of integrated management strategies. Among the biocontrol agents the entomopathogenic fungi (EPF) can able to give safe and effective control of insect pests. They have a wide host range and therefore some of the virulent entomopathogenic fungal isolates have been commercially produced as biocontrol agents for many sucking and chewing insects (Hajek et al., 2017).

Biocontrol methods based on EPF are not only relying on host pathogen interaction, but also on the environment to which they are exposed. The different environment factors including, temperature, relative humidity (RH), light, air, nutrient content and host physiological status will influence the pathogenicity of entomopathogenic fungi (Padmini and Padmaja, 2010). Among the various environmental factors, temperature and humidity are the most important affecting survival and virulence of EPF (Bugeme et al., 2008). Many studies have reported the effect of temperature on germination, radial growth and sporulation of EPF and the results revealed that optimum temperature for most of the entomopathogenic fungal isolates ranging between 20-30°C, depending on the isolate (Rangel et al., 2005; Fargues et al., 1996). For field application, the most virulent isolates should also be tolerant to different temperature ranges where the targetpest is more dominant (Fernandes et al., 2007; Braga et al., 2007). As a result, these environmental factors should be taken into consideration while assessing EPF’s commercial potential. Taking into account the aforementioned conditions, the present study aimed at determining the effects of various temperatures on germination, radial growth and sporulation of different entomopathogenic fungal isolates of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium flavoviride Gams and Rozsypal var. minus and on their pathogenicity to two spotted spider mite, T. urticae.

Source of entomopathogenic fungal isolates
 
Five entomopathogenic fungal isolates viz., B. bassiana (Bb 111), B. bassiana (Bb 112), B. bassiana (Bb 113), B. bassiana  (Bb 114) and M. flavoviride var. minus were obtained during the survey in the year 2018. Spore suspensions of 1 x 106 spores ml^-1 of all the test isolate were spread-plated on SMA+Y (Sabouraud Maltose Agar enriched with Yeast extract) Petri dishes separately for getting culture with similar age for the study and incubated at the temperature 25±1°C for seven days.
 
Mass culturing of red spider mite, T. urticae
 
The spider mite population used for various experiments was mass cultured in the Insectary, Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore. Bhendi leaves severely infested with the red spider mite under field conditions were collected and placed on the potted plants to begin the base culture of mites. When plants suffered heavy infestations, new colonies were initiated on newly grown potted plants (Saranya, 2012).
 
Effect of temperature on radial growth and sporulation
 
To study the impact of temperature on radial growth and sporulation, three days old entomopathogenic fungal culture isolates were used. From each isolate, 5mm mycelial discs was cut with the help of sterilized cork borer and transferred to the centre of fresh SMA+Y plates. Inoculated plates were incubated at test temperatures viz., 20°C, 25°C, 30°C and 35°C and allowed for 15 days to attain maximum vegetative growth. Each treatment was replicated three times. Fully sporulated cultures were utilized to determine the spore count using Neubauer hemocytometer.
 
Effect of temperature on germination

Ten μl of an aqueous spore suspension in 0.05 per cent Tween 80 containing 1 x 106 spores ml^-1 was spread on a cavity slide coated with thin film of SMA+Y medium. The inoculated slides were kept in Petri dishes lined with blotting paper moistened with sterile water. Petri dishes were sealed and placed in incubators set at four test temperatures (20°C, 25°C, 30°C and 35°C). Three replicates were maintained for each isolate. The slides were observed under a compound microscope after 24 h of inoculation and the spores were considered as germinated when the germ tube was at least twice the diameter of the spore. Percentage germination was then determined by counting 100 spores for each plate at 400X magnification (Storey and Gardner, 1986).
 
Virulence of B. bassiana isolates grown at different temperatures against T. urticae
 
Laboratory bioassays on T. urticae were carried out to assess the virulence of different entomopathogenic fungal isolates subjected to different temperature. For all the fungal isolates, test temperatures subjected, 21 days old test cultures were chosen for the bioassay studies. Bioassays were performed, using a leaf dip method, following the procedures described by Xu et al., (2002). Leaves from bhendi plants were collected and were cut into circular disc of 50 mm diameter size and placed on 1.5 per cent agar in a Petri dish. Thirty mites were collected individually from base culture were released in each leaf disc. All fungal strains were prepared at a standard concentration of 1 x 106 spores ml^-1 in 0.05% Tween 80. Observations on the mortality were recorded daily up to seven days. The percent mortality in each treatment was calculated and corrected by Abbott’s formula (Abbott 1925).
 
Statistical analysis
 
The data obtained were subjected to square root (X+0.5) transformation and the analysis of variance in experiments was carried out in AGRES and the means were separated by least significant difference (LSD) available in the package.

Effect of temperature on radial growth
 
Influence of different temperature regimes viz., 20°C, 25°C, 30°C and 35°C on the growth of different fungal isolates was studied. Results indicated that all the isolates had highest radial growth at 25°C. While, minimum radial growth was observed at 20°C, 30°C, 35°C for all the isolates except M. flavoviride var. minus, this ceased to grow at 35°C. At 25°C, B. bassiana (Bb 111) isolate showed a maximum radial growth of 84.67 mm. The isolate Bb 111 showed more than 70 mm radial growth at 20°C and 25°C (Table 1). Maximum growth at 30°C and 35°C was recorded with the isolates Bb 112 (56.67 and 26.67 mm) and Bb 111 (44.33 and 22.33 mm) after 14 days of inoculation (Fig 1). 





Environmental temperature and relative humidity (RH) are known to affect spore germination, colony growth and host infection capacity of the fungi (Tanada and Kaya, 1993; Feng et al., 1994; Roberts and St. Leger, 2004). Appropriate temperature and high RH are usually crucial for the successful infection of the fungal agents (Milner, 1997; Luz and Fargues, 1999). Temperature dependent growth and infectivity has been demonstrated for many hyphomycetous fungi including B. bassiana and M. anisopliae (Walstad et al., 1970; Fargues et al., 1997; Ekesi et al., 1999; Milner et al., 2003).

The radial growth was highly influenced by incubation at 25°C in all the tested strains. Over all, the cumulative growth revealed the highest radial growth at 25°C by Bb 111 (84.67 mm), Bb 112 (73.33 mm) and Bb 114 (65.67 mm) which was significantly higher than the growth at other tested temperatures. Similarly, when the incubation temperature was lower (20°C) there was corresponding decrease in growth. It has been reported by several authors that several fungi have different temperature optima for their growth (Duncan, 1973). The temperature limits for growth range were between 5°C and 35°C and the optima fall between 20° and 30°C (Roberts and Yendol, 1971; Zimmermann, 1982). This result coincides with the findings of Fargues et al., (1992) and Dimbi et al., (2004) who reported that the optimal temperature for growth was 25°C for the isolates of M. anisopliae. Taylor and Khan (2010) reported that the optimum temperature for growth for all fungal isolates appears to lie between 25 to 30°C.

Effect of temperature on sporulation
 
Temperature significantly affected the sporulation of the entomopathogenic fungal isolates. The optimum temperature for sporulation was found to be 25°C for all the five entomofungal strains tested except M. flavoviride var. minus. The maximum spore production was observed with Bb 112 at 25°C (1.63 x 106 spores ml^-1) followed by Bb 111 (1.45 x 106 spores ml^-1), Bb 114 (1.20 x 106 spores ml^-1) and Bb 113 (1.15 x 106 spores ml^-1 (Table 2). The isolate M. flavoviride var. minus did not sporulate at all the temperature (20°C, 25°C, 30°C and 35°C) tested.



Similar results for tropical isolates of entomopathogenic fungi were documented by several authors (Davidson et al., 2003; Yeo et al., 2003; Rodriguez et al., 2009). In our study, the mmaximum spore production was observed with Bb 112 at 25°C (1.63 x 106 spores ml^-1) followed by Bb 111 (1.45 x 106 spores ml^-1) and Bb 114 (1.20 x 106 spores ml^-1). Similar results were reported by Tefera and Pringle (2003) on M. anisopliae and Arthurs and Thomas (2001) on M. anisopliae var. acridium.
 
Effect of temperature on conidial germination
 
There was a significant effect of temperature on germination of conidia at 24 h post inoculation (Fig 2). The optimum temperature for germination of all the strains was found to be at 25°C (Table 3). The maximum germination was observed at 25°C which varied between 1.67 and 95.67 per cent. Germination at 35°C was low (0 to 36.67%) for all the strains. Among the isolates tested, Bb 112 showed maximum germination at 20°C (82.00%), 25°C (95.67%) and 35°C (36.67%). At 30°C maximum germination was observed in the culture Bb 114 (87.67%).





In the present investigation, the optimum temperature was found to be 25°C for all the observed parameters among the isolates. The germination of Bb 112 isolate was 95.67 per cent at 25°C and this is in line with the findings of Tefera and Pringle (2003) who observed more than 80 per cent germination of B. bassiana isolates when incubated at 25°C. Glare and Milner (1991) reported that most of the isolates of B. bassiana are mesophilic with an optimum temperature requirement of 25 to 30°C. Ekesi et al., (1999) also reported that B. bassiana strain Bb-01 had an optimal germination at 25°C. Germination at 35°C was low (<37.00%) for all the strains in the present study is in tune with the findings of Walstad et al., (1970), Ferron et al., (1991) and Dimbi et al., (2004).
 
Virulence of B. bassiana isolates grown at different temperatures against T. urticae
 
There was a significant difference in the virulence grown at 25°C between isolates against T. urticae. All isolates induced more than 50 per cent mortality in seven days. The isolate Bb 112 caused higher mortality of 97.57 per cent followed by Bb 111 (94.18%) (Fig 3) and M. flavoviride var. minus (91.65%) (Table 4). At 30°C Bb 112 and Bb 111 were more effective than other isolates. The isolates were comparatively less virulent were grown at 20°C and 35°C.





Generally, the entomopathogenic fungal isolates were all pathogenic to the host when grown at all temperatures (20°C, 25°C and 30°C) except at 35°C. More than 90 per cent mortality was observed with the isolates grown at 25°C. The results are in accordance with the findings of Bugeme et al., (2008) against T.  urticae. Similar results have been reported in the legume flower thrips Megalurothrips sjostedti (Trybom) (Ekesi et al., 1999), second instar Chilo partellus (Swinhoe) larvae, three species of African tephritid fruit flies Ceratitis capitata (Weidemann), C. cosyra (Walker) and C. fasciventris (Bezzi) (Dimbi et al., 2004) and Coptotermes formosanus (Sun et al., 2003).

The temperature not only affects the physiology of the fungus but also the ability of fungus to infect the host. Identifying a fungal strain with a broad temperature range is therefore necessary for a rational approach to the management of insect pests in field. In the present study the isolates Bb 111 and Bb 112 were found promising in all the tested parameter with varying temperatures.