Abstract: Studies were carried out in Plant Protection Research Institute, Sharkia branch during 2012-2013 years to determine the effects of different temperatures (10, 15, 20, 25, 30, 35 and 40°C), pH values (2, 4, 6, 7, 8, 10 and 12) and incubation periods (2, 4, 6, 7, 8, 10, 12 and 14 day) on Isaria fumosorosea Wize mycelial growth rates as a biological control agent of Planococcus citri (Risso). The toxicity of I. fumosorosea spores suspension on citrus mealybug, P. citri under laboratory conditions was studied. The optimal environmental conditions for the mycelial growth rate of I. fumosorosea were obtained at 25°C, in pH 5.8, for 7 days incubation period. The LC50 value was 5x106 spores mL-1 and LC90 was 4x1013 spores mL-1.
INTRODUCTION
The entomopathogenic fungi, Isaria farinosa and Isaria fumosorosea were known as Paecilomyces farinosus and Paecilomyces fumosoroseus, for more than 30 years. Both fungi have a worldwide distribution and a relatively wide host range. While, I. farinosa currently is of minor importance in research and as biocontrol agent, I. fumosorosea is regarded as a species complex and various strains are successfully used for biocontrol of several pest insects (Zimmermann, 2008). Paecilomyces spp., a common insect borne filamentous fungus belongs to Hyphomycetes of Deuteromycota and has been reported to cause diseases in a wide species of insects, occasionally resulting in natural epizootics (Altre et al., 1999; Cantone and Vandenberg, 1999; Lacey et al., 1999; Nam et al., 2000). Panyasiri et al. (2007) founded that five strains of I. fumosorosea isolated from a Coleopteran, an unkown host and white flies, were highly active against thrips, Ceratothripoides claratris (80-93% mortality), moderate activity against Bemisia tabaci (37-77% mortality) and less active against mealybug, Pseudococcus cyrptus (10-43% mortality). Also, Demirci et al. (2011) reported that the entomopathogen I. farinose may be used as a biocontrol agent against citrus mealybug, Planococcus citri.
Fungal spores are living organisms and their viability diminishes with time depending on environmental conditions. Temperature is considered as one of the important factors affecting the natural activity of parasitic fungi. The rapidity of mycelial development and evaluation of infection depend on temperature. In general, the optimum growth and germination rates on artificial media vary around 25°C for P. fumosoroseus (MacLeod, 1963; Kalvish, 1974; Ekbom, 1979; Fang et al., 1985; Moore et al., 2000). Paecilomyces fumosoroseus mycelial growth has been expedited gradually in proportion to the rise of temperature and was the most suitable at 25°C. Even though the mycelial growth of P. fumosoroseus was favorable at the range of 20-25°C and had been expedited in proportion to the rise of temperature, the mycelial growth appeared to be suppressed at the temperature higher than 30°C. Similarly, there was slow growth at 15 and 35°C (Shim et al., 2003b).
The pH is considered as one of the important factors affecting the natural activity of fungi. Choi et al. (1999) reported that mycelial growth of P. japonica was optimal at pH 7. Shim et al. (2003a) also reported that P. sinclairii showed maximal mycelial growth at pH 8. The mycelial growth and density of P. fumosoroseus was almost identical in the range of pH 6-9. The pH values suitable for a favorable growth of P. fumosoroseus were obtained in the range of pH 6-9 (Shim et al., 2003b).
Incubation period considered as one of the important factors affecting the natural activity of parasitic fungi. Ghareeb (2009) indicated that fungal growth of Paecilomyces violaceae increased rapidly during the linear growth phase. The optimum biomass production by tested fungi was achieved at the end of 5 days of growth periods. However, biomass yield gradually decreased during the longer period of the culture (7-12 days). Growth rate was gradually dependant on the duration of fermentation period.
The present study aimed to determine the effects of some abiotic factors such as temperature, pH values and incubation periods on growth rate of entomopathogenic fungi, Isaria fumosorosea and its effectiveness on citrus mealybug, Planococcus citri under laboratory conditions.
MATERIALS AND METHODS
Studies were carried out in Plant Protection Research Institute, Sharkia Branch during 2012-2013 years to determine the effects of some abiotic factors such as temperature, pH values and incubation periods on growth rates of entomopathogenic fungi, Isaria fumosorosea and the pathogenisty against citrus mealybug, P. citri.
Isolation, identification and maintenance of fungal culture stock: Isaria fumosorosea was isolated from naturally infected Ceroplastes floridensis (Soft scale insect) on lemon trees at Sharkia Governorate in 2012 and identified using ribosomal DNA sequences data according to Abd-Elsalam et al. (2003). The fungal isolates used in present study were descendant of pure single slant culture. The stock culture of fungal isolates were maintained on Czapek-Dox’s and potato dextrose then stored at 4.0°C with transfer at monthly intervals slant (Smith and Onions, 1983).
Effects of some abiotic factors: Each treatment was carried out in triplicates and the results obtained throughout this study were the arithmetic average of at least two experiments.
Temperature: In this experiment, Sterilized Czapek-Dox’s liquid medium adjusted at pH 6 were inoculated with equal amounts of the fungal spores and incubated at the following temperature 10, 15, 20, 25, 30, 35 and 40°C. At the end of incubation period after 7 days, cultures were harvested for determining the mycelial dry weights.
pH values: The basal medium was adjusted to following pH values, 2, 4, 6, 7, 8, 10 and 12 by addition of varying amounts of 1 N HCl and NaOH using pH electrode. The inoculated flasks were incubated at 25°C for 7 days after which, mycelial dry weights were determined.
Incubation periods: Sterilized Czapek-Dox’s liquid media was inoculated with culture of I. fumosorosea, incubated at 25°C and the experiment was extended for 14 days. Dry weight was determined respectively every 48 h.
Preparation of inocula: Inoculum was prepared by agitating a slant of the fungal isolates (7 days old fungal culture) with 10 mL sterile distilled water using an inoculated needle. One milliliter of the spores suspension was then used as standard inocula.
Cultivation: Erlenmeyer conical flasks (250 mL capacity) for each treatment, each one containing 50 mL of fermented medium, were used in the present study. The pH of the medium adjusted to 6. The flasks were plugged with cotton wool and sterilized at 121°C for 20 min. Each flask, after being cooled was inoculated with 1 mL spore suspension (standard inoculum) under aseptic conditions. The culture flasks were then incubated at 25°C in incubator.
Determination of mycelial dry weight: At the end of the required incubation period, the culture flasks were filtered through preweighted Whatman No. 1 filter papers. The filter papers were washed twice with distilled water, dried in an electric oven at 80°C and left in desiccator to attain room temperature. The filter papers were then weighted at regular intervals till the two successive weights were the same.
Laboratory evaluation
Culture of Planococcus citri: The citrus mealybug, P. citri was reared on Squash fruit (Cucurbita pepo) under laboratory conditions 25±1°C, 65±5 RH% and 12 h photoperiod. Baby Sun Rose leaves, Aptenia cordifolia were preferred for the laboratory evaluation in order that its leaves can survive for a long period after detached. Planococcus citri ovisacs were taken by a fine brush and located on A. cordifolia leaves. The leaves were incubated under the same laboratory conditions 25±1°C, 65±5 RH% and 12 h photoperiod. After 48 h from ovisac hatching 5 leaves were puked up and put in a plastic petri plates on filter papers that saturated daily with water.
Fungal inocula: Spores of fungal isolates were harvested by rinsing with sterilized 0.005% Tween 80 from 7 day old culture (PDA media grown at 25±1°C for I. fumosorosea isolates). The suspensions were filtered through cheesecloth to reduce mycelium clumping. The spores were counted in the suspensions using a haemocytometer. The concentrations were adjusted to 1x105, 1x106, 1x107 and 1x108.
Experimental design: Experimental studies were applied on three replicates for each concentration and the same for control. Each replicate contains 5 leaves of A. cordifolia every leaf has 5 individuals of P. citri larval instar. Leaves sprayed with 2 mL of spores suspension and the control was treated with 2 mL of 0.005% Tween 80 only. The treatments and control were incubated for 7 days under laboratory conditions 25±1°C, 65±5 RH% and 12 h photoperiod. Larval mortality was observed after 1, 3, 5 and 7 days. The LC50, LC90 and slope values were calculated after 7 days according to Finny (1971).
RESULTS AND DISCUSSION
Effects of temperature: Data given in Table 1 showed that there was gradual increase in mycelial growth rates of I. fumosorosea under temperatures of 10, 15, 20 and 25°C with values of 0.02, 0.06, 0.23 and 0.47 g/100 mL, respectively. After that mycelial growth rates were decreased with values of 0.45, 0.14 and 0.05 under temperatures of 30, 35 and 40°C. The optimal temperature for mycelial growth was obtained at 25°C with value of 0.47 g/100 mL. This result was agreed with those given by MacLeod (1963), Kalvish (1974), Ekbom (1979), Fang et al. (1985), Moore et al. (2000) and Shim et al. (2003b) who reported that P. fumosoroseus mycelial growth has been expedited gradually in proportion to the rise of temperature and the optimum temperature for growth and sporulation were ranged between 20-25°C.
Effects of pH values: Data given in Table 2, clarified that there were seven levels of pH that were adjusted at 2, 4, 6, 7, 8, 10 and 12. The mycelial dry weight of I. fumosorosea reached values of 0.07, 0.17, 0.09, 0.10, 0.13, 0.07 and 0.07 g/100 mL, respectively. The suitable pH final value for mycelial dry weight was obtained at pH 5.8 with value of 0.17 g/100 mL. This result was agreed with this given by Shim et al. (2003b) who reported that the suitable pH values for a favorable growth of P. fumosoroseus were obtained in the range of pH 6-9.
Effects of incubation periods: Data given in Table 3 showed that the optimum incubation period for mycelial growth of I. fumosorosea was obtained after 7 days of incubation with value of 0.45 g/100 mL.
This result was agreed with Ghareeb (2009) who reported that P. violaceae biomass increased rapidly during the linear growth phase. The optimum biomass production by tested fungi was achieved at the end of 5 days of growth periods. The yield gradually decreased during the longer period of the culture (7-12 days). Growth rate was gradually dependant on the duration of fermentation period.
| Table 1: | Effect of different temperatures on growth rates of Isaria fumosorosea after 7 days |
| Table 2: | Effect of different levels of pH values on growth rates of Isaria fumosorosea after 7 days |
| Table 3: | Effect of different incubation periods on growth rates of Isaria fumosorosea under laboratory conditions 25±1°C,65%±5 RH% and 12 h photoperiod |
| Table 4: | Mortality percentages of larval instars of Planococcus citri after application with different concentrations of Isaria fumosorosea spores suspension under laboratory conditions (25±1°C, 65%±5 RH% and 12 h photoperiod) |
| Table 5: | Lethal concentration (LC25-99) of Isaria fumosorosea spores per mL after 7 days of application against larval instar of Planococcus citri under laboratory conditions 25±1°C, 65%±5 RH% and 12 h photoperiod |
Laboratory evaluation: Data given in Table 4 showed the efficacy of I. fumosorosea spores suspension on larval instars of citrus mealybug, P. citri, after application with different concentrations of I. fumosorosea under laboratory conditions 25±1°C, 65±5 RH% and 12 h photoperiod. The concentrations were adjusted to 1x105, 1x106, 1x107 and 1x108 spores mL-1. Mortality percentages after 7 days of application showed 36.00, 48.00, 49.33 and 60.00%, respectively.
Data given in Table 5 clarified the LC50 and LC90 values of I. fumosorosea spores/mL after 7 days of application on larval instars of citrus mealybug, P. citri. The obtained results revealed that LC50: 5x106 spores mL-1 and LC90: 4x1013 spores mL-1 (Fig. 1).
| Fig. 1: | Concentration mortality probit line of Isaria fumosorosea spores per mL on larval instars of Planococcus citri under laboratory conditions 25±1°C, 65±5% RH and 12 h photoperiod after 7 days |
This result was agreed with that of Panyasiri et al. (2007) who found that I. fumosorosea has moderate activity against B. tabaci (37-77% mortality) and less active against mealybug, P. cyrptus (10-43% mortality) and Demirci et al. (2011) who recorded that entomopathogenic fungi, I. farinosa used as biocontrol agent against mealybug, P. citri causing 89.39% mortality ovisac, 84.07% mortality in second larval stage, 84.53% mortality in adult females and 78.71% mortality in first larval stage at 1x108 conidia mL-1 inoculm concentration.