Susceptibility of Tetranychus urticae Koch . ( Acari : Tetranychidae ) to Isolates of Entomopathogenic Fungus Beauveria bassiana

Bioassays with five isolates of the entomopathogenic fungus Beauveria bassiana (Bals.Criv.) Vuillemin were conducted under laboratory conditions with a goal to estimate their virulence to the two-spotted spider mite Tetranychus urticae Koch. (Acari: Tetranychidae). Common bean plants with mites were treated by spraying conidial suspensions of isolates at concentrations of 106, 107 and 108 conidia/ml. Lethal effects of the fungal isolates were evaluated as percentages of cumulative daily mortality due to mycoses, corrected for mortality in the control variant. Virulence of the isolates was estimated based on values of the median lethal time (LT50) calculated by probit analysis for the variants treated with conidial suspensions at the concentration of 106 conidia/ml. The two-spotted spider mite was found susceptible to the examined isolates of the entomopathogenic fungus B. bassiana. Mycosis caused to T. urticae by the B. bassiana isolates 444 Bb and 445 Bb had fast lethal effect after treatment with conidial suspensions even at the concentration of 106 conidia/ ml. The mean mortality values of host individuals were 83.78 ± 3.62% and 68.49 ± 4.28% on the first day, respectively, and up to 100% in both variants on the fourth day. The isolates 426 Bb, 444 Bb and 445 Bb of B. bassiana were highly virulent to two-spotted spider mites with values of the median lethal time varied within overlapped narrow confidence intervals from 0.122 to 1.084 days (average value 0.162 days), from 0.117 to 1.398 days (average value 0.146 days) and from 0.106 to 1.162 days (average value 0.131 days), respectively. Significant differences regarding virulence of the three isolates at p-level < 0.05 could not been proved. The other two examined isolates were distinctly less virulent to T. urticae than these three B. bassiana isolates.


INTRODUCTION
Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan and polyphagous species with great economic impotance for crops in greenhouses and in the field.Conventional control of this pest includes acaricide treatments that could lead to undesirable side effects, such as death of non-target organisms (e.g.predators), development of pesticide-resistant races of two-spotted spider mite and residue concerns.Methods of integrated pest management are consistent with the consumers' demand to reduce health and environment risks.Screening for eligible biocontrol agents is a step in developing new or improving existing environment-friendly strategies offering an alternative to conventional pest control.
Opportunities to exploit entomopathogenic fungi for biological control of mites were summarized by Chandler et al. (2000), Alves et al. (2002) and Maniania et al. (2008).Maniania et al. (2008), reviewing the use of fungal pathogens in inundative, conservation and conventional biological control of T. urticae and T. evansi, concluded that there is a great potential for use of entomopathogenic fungi in conservation and conventional biological control programs.Successful use of entmopathogenic fungi as microbial control agents of mites will ultimately depend on how well the strains are selected.For development of mycoacaricides based on entomopathogenic fungi in the Ascomycota, order Hypocreales, screening for more efficient strains is still necessary.
The current study was designed to determine the virulence of five Bulgarian isolates of the entomopathogenic fungus B. bassiana to the two-spotted spider mite T. urticae.

MATERIAL AND METHODS
Five fungal isolates of B. bassiana, applied in bioassays, were provided from a collection of entomopathogenic fungi maintained at the Department of Biological and Integrated Pest Control (Plant Protection Institute, Bulgaria).They were initially isolated into pure cultures from dead insects collected from natural pest populations found in different regions of Bulgaria.The isolate marked 417 Bb was obtained from a larva of Hedya nubiferana (Haw.)(Lepidoptera: Tortricidae) found in an apple orchard near Sofia in 2006; the isolates marked 444 Bb, 445 Bb and 447 Bb from larvae of Cydia pomonella L. (Lepidoptera: Tortricidae) found in apple orchards near the town of Plovdiv in 2007, and the isolate marked 426 Bb from a dead adult of Ips sexdentatus Boer.(Coleoptera: Scolytidae) found in pine bark in the Malashevska Mountain in 2006.
In order to prepare conidial suspensions for bioassay, fungal isolates were cultured on SDAY (Sabouraud dextrose agar with yeast extract) in tubes at 25 ± 1 o C for 15 days.Conidia of each isolate were harvested by washing down with 10 ml sterile water per tube followed by decimal dilutions of the obtained conidial suspensions and determination of their concentrations by haemocytometer.
Bioassays with two-spotted spider mites were conducted under laboratory conditions (23 ± 1 o C, photoperiod 12:12 L:D) with a host population reared on common bean plants (Phaseolus vulgaris L.) in pots.The plants with mites were treated by spraying the prepared conidial suspensions at the following concentrations: from 0.906 x 10 6 to 0.906 x 10 8 conidia/ml in variants with the fungal isolate 417 Bb; from 0.838 x 10 6 to 0.838 x 10 8 conidia/ml in variants with the isolate 426 Bb; from 0.590 x 10 6 to 0.590 x 10 8 conidia/ml in variants with 444 Bb; from 1.300 x 10 6 to 1.300 x 10 8 conidia/ml in variants with 445 Bb, and from 1.040 x 10 6 to 1.040 x 10 8 conidia/ml in variants with 447 Bb.
Control variants were treated with water.The experiments were proved in 3 replicates.Mite mortality was checked daily during five days.Leaves with dead mites were placed in a humid chamber to allow reproductive structures of the fungus to develop and to confirm that mycosis was the reason of death.Smears prepared from dead mites were stained by methylen blue and were observed under light microscope.
Lethal effects of the isolates were evaluated as percentages of cumulative daily mortality due to mycoses, corrected for mortality in the control variant according to Abbott's formula (1925).Virulence of the isolates was estimated based on median lethal time (LT 50 ) calculated by probit analysis (Finney, 1971) for the variants treated with conidial suspensions at the concentration of 10 6 conidia/ml.Confidence intervals of varying LT 50 values were calculated at p-level < 0.05.

RESULTS AND DISCUSSION
Bioassay with five isolates of the entomopathogenic fungus B. bassiana showed that two-spotted spider mites with mycosis became red and later lost their turgor.Investigation of smears of mites' cadavers confirmed mycosis as the cause of death in variants treated with conidial suspensions of the fungal isolates.
The bioassay results showed that two-spotted spider mites were susceptible to the examined isolates of the entomopathogenic fungus B. bassiana.As shown in Figure 1, mites in the variants treated with conidial suspensions of the isolates 444 Bb, 445 Bb and 426 Bb at concentration of 10 6 conidia/ml developed mycosis with fast lethal effect.The mean mortality values for individuals on the first day were high -83.78± 3.62%, 68.49 ± 4.28% and 56.88 ± 7.06%, respectively.Infections due to isolates 417 Bb and 447 Bb were less lethal -19.30 ± 5.71% and 5.66 ± 0.76% mortality in the variants treated with conidial suspensions at concentration of 10 6 conidia/ml.On the second day, mortality in the variants with isolates 444 Bb and 445 Bb increased to 91.36 ± 1.18% and 84.24 ± 0.53%, respectively, and than to 99.21 ± 0.79% and 94.99 ± 1.30% on the third day, and to 100% in both variants on the fourth day.The established mortality from fungal disease in the variants with isolates 447 Bb and 426 Bb was as follows: 57.05 ± 0.65% and 64.15 ± 6.03%, respectively, on the second day; 78.66 ± 4.96% and 80.88 ± 7.24% on the third day, and 97.60 ± 0.31% and 82.79 ± 7.01% on the fourth day.On the fifth day, lethal effect reached 100% in the variant with 447 Bb, and 84.87 ± 19.17% in the variant with 426 Bb.Mycosis caused by the isolate 417 Bb of B. bassiana had a less lethal effect -68.89 ± 7.40% calculated for the fifth post-treatment day.
In our view, the fast lethal effect of the mycoses to T. urticae was due to toxic secondary metabolites and the pigment oosporein produced by fungal isolates.Growing the examined isolates 417 Bb, 426 Bb, 444 Bb, 445 Bb and 447 Bb of B. bassiana a red pigmentation of the media was also noticed.
The main characteristic that has an important role in the virulence of entomopathogenic fungi strains from the genera Beauveria and Metarhizium is the production of enzymes necessary for penetration through arthropod cuticle.The extracellular proteases are considered an important virulence factor in insect disease processes (St. Leger et al., 1986a, 1986b;1988;Draganova, 1988;Bidochka and Khachatourians, 1990, 1994a, 1994b;Gupta et al., 1992;St. Leger, 1995).The extracellular chymoelastase (Pr 1) was noticed as the most important enzyme for overcoming the resistance of the insect integument (St. Leger et al., 1988;Bidochka andKhachatourians, 1994a, 1994b).Besides enzymes, many isolates of Beauveria species have been found to produce toxic compounds after invasion of the host haemolymph (Roberts, 1981).Some of the compounds were beauvericin, beauverolides, bassianolide, isarolides, pigments (bassianin, tenellin, oosporein) and oxalic acid (Roberts, 1981;Eyal et al., 1994;Strasser et al., 1998Strasser et al., , 2000)).Production of toxic metabolites was a step in mycosis development followed by death of the host.In the present bioassay, treatment with higher concentrations of conidial suspensions (10 7 and 10 8 conidia/ml) was found to result in higher mortality of the host but while lethal effect in more of the variants could be distinguished on the first day after treatment, significant differences among the mortalities in different variants could not be proved after the third day and later (Figure 2 and Figure 3).
Virulence of the examined isolates of B. bassiana to T. urticae was estimated based on values of the median lethal time (LT 50 ) calculated for the variants treated with conidial suspensions at the concentration of 10 6 conidia/ml (Table 1).The isolates 426 Bb, 444 Bb and 445 Bb were highly virulent to the two-spotted spider mite.Their values of the median lethal time varied within overlapped confidence intervals from 0.122 to 1.084 days (average value 0.162 days), from 0.117 to 1.398 days (average value 0.146 days) and from 0.106 to 1.162 days (average value 0.131 days), respectively.Significant differences in the virulence of isolates 426 Bb, 444 Bb and 445 Bb (at p-level < 0.05) could not been proved.The isolates 417 Bb and 447 Bb were less virulent to T. urticae.The calculated p-level < 0.05 values of LT 50 for these variants were within overlapped confidence intervals from 2.864 to 3.258 days (average 3.054 days) and from 3.138 to 3.174 days (average 3.156 days), respectively.So the isolates 417 Bb and 447 Bb could not be distinguished for their virulence to T. urticae based on median lethal time but significant differences were proved between the two isolates and the other examined isolates.Calculated confidence intervals for the fifth fungal isolate were narrow, indicating an equalized susceptibility within the treated population of T. urticae to the studied B. bassiana isolates.
In conclusion, the two-spotted spider mite was found to be susceptible to the examined isolates of the entomopathogenic fungus B. bassiana.Mycosis caused to T. urticae by the isolates 444 Bb and 445 Bb had fast lethal effect after treatment with conidial suspensions even at the concentration of 10 6 conidia/ml.The mean mortality values for host individuals on the first day were 83.78 ± 3.62% and 68.49 ± 4.28%, respectively, and on the fourth day up to 100% in both variants.
The isolates 426 Bb, 444 Bb and 445 Bb were highly virulent to two-spotted spider mites with values of the median lethal time varied within overlapped narrow confidence intervals from 0.122 to 1.084 days, from 0.117 to 1.398 days and from 0.106 to 1.162 days, respectively.Significant differences in the virulence of the three isolates at p-level < 0.05 could not been proved, unlike virulence between the isolates 417 Bb and 447 Bb.

Table 1 .
Virulence of isolates of the entomopathogenic fungus Beauveria bassiana to two spotted spider mite Tetranychus urticae