Virulence of Verticillium Iecanii (Zimm.) Viegas Subcultures Grown on an Artificial Medium or its Natural Host Icerya Seychellarum (Hemiptera: Monophlebidae)

INTRODUCTION

      Virulence of the entomopathogenic fungi is affected by repeated successive subculturing in artificial media or passages through its insect hosts. Through successive subculturing on artificial media Constancy of fungal virulence is a desirable for biocontrol agents' production (Brownbridge et al., 2001; Vandenberg and Cantone, 2004). Enhancement of entomopathogenic fungi virulence following repeated subculturing in artificial media or passage through insect hosts has been reported (Kawakami, 1960; Schaerffenberg, 1964; Fargues and Robert, 1983). Some studies have reported that successive cultures in artificial media cause attenuation in fungal virulence. However, other studies proved no decline in virulence of fungi, subcultured in artificial media (Ansari and Butt, 2011; Brownbridge et al., 2001; Vandenberg and Cantone, 2004). Single passage of these fungi through a suitable host can restore or increase the virulence.While some studies reported that virulence needs two or more successive passages to be increeased (Butt et al., 2006). The effect of repeated subculturing in vitro and vivo passaging on viability, morphological characteristics and virulence, varies within entomopathogenic fungi strains (Cooper and Sweeney 1986; Guedes-Frazzon et al., 2000). Several studies suggested that the effect of repeated in vitro and has an impact on virulence in vivo passaging on the virulence of entomopathogenic fungi(Hajek et al., 1990) For some unknown reason, virulence may be temporarily restored in some subcultures but the overall trend is a decline.Asghar (2013) investigated the effects of repeated subculturing of Beauveria bassiana and Metarhizium anisopliae in vitro and passages through insects on their virulence against Uvarovistia zebra. The virulence of both fungi was reduced after four subcultures in Potato Dextrose Agar (PDA) media, but this reduction was not quite significant for B. bassiana. Attenuated fungi obtained from the fourth subculturing were passaged through 3^rd instar nymphs of Uvarovistia zebra. The insect passage was repeated two times and the fungi virulence was evaluated by calculating mortality percent. Following passage there was a small, but non-significant increase in the fungi virulence. This study aimed to determine the virulence of subcultures of the entomopathogenic fungus Verticillium lecanii derived either form an artificial diet or from rearing on a natural insect-host (Icerya seychellarum).

MATERIALS AND METHODS

The tested fungus and insects : The entomopathogenic fungus Verticillium lecanii (Zimmerman) viegas which was reclassified by Zare and Gams (2001) as Lecanicillium lecanii with a high virulent pathogenicity against scale insects and mealy bugs it was originally isolated from Alaska (USA and EMCC Number: 919^TM). The culture was obtained from Egypt microbial culture collection (Ain Shams Univ., Egypt). The adult individuals of the mealy bug Icerya seychellarum were reared on citrus leaves which have been replaced in Petri dish surrounded with a wet tissue paper to keep it fresh as insect host medium.

Fungal maintenance and conidial preparations:  Medium cultures were maintained on sabouraud dextrose agar (SDA)media (10g mycopeptone, 40 g dextrose, 15 g agar and 1000 ml distilled water).  For the fungal growth, malt agar with 0.1% yeast extract were added to SDA medium and kept for 10 days at 25°C. Spores were harvested with an aqueous solution of 0.05% Triton -100^®. The spore suspension was filtered through several layers of cheese cloth to remove mycelial mats. The concentration of spores in the final suspension was determined using a haemocytometer and adjusted for bioassay was by dilution with 0.05% Triton -100 to a final stock concentration of 1.7X10⁸spores/ml. Thisprocedure was done according to (Mehta et al, 2012).

Conidial subculturing on artificial media : For subculturing and repeated in vitro spore transfers, the spores were harvested from the surface of 10 days old culture by scraping with a loop and subcultured to fresh molasses yeast broth (MYB) (30g molasses,5g yeast and 1000 ml distilled water).  Cultured fungi were incubated at 28°C. This multi-spore in vitro transfer was repeated up to 4 times. Before subculturing, a suspension of harvested spores was prepared to be used for bioassay and inoculate other insects. Thisprocedure was done according to (Asghar,2013).

Conidial passaging through insect: For the in vivo passage of the fungus maintained in the insect-host, spores of the 1^st, 2^nd, 3^rd, 4^th and 5^th in vitro subcultures were recovered from Petri dishes and suspended in sterile distilled water. Spore concentration of the suspension was adjusted to 1.7×10⁸ spores/ml. Adult individuals of the mealy bug Icerya seychellarum were dipped in the spore's suspension (1.7×10⁸spores/ml) containing 0.02% Tween 80^® solution and placed individually in a small plastic container. Inoculated insects were maintained on a lettuce until response (mortality) occurred. Dead insects were cleaned with cotton wool soaked with 70% ethanol and incubated at 28°C with 80-90% R.H to stimulate sporulation of the fungi. The spores from cadavers were harvested by scraping with a pointed needle and suspended in sterile distilled water to inoculate other insects. This insect passage was repeated and virulence of the fungi was evaluated by mortality through a bioassay. Thisprocedure was done according to (Asghar,2013).

Rearing the tested insects: The immature individuals of the used mealy bug, Icerya seychellarum were reared on young citrus (orange) trees (1 year old). The trees were individually infested with 60 immature mealy bugs. Four weeks after the initial infestation, the settled adults of I. seychellarum were observed and used for bioassay measuring the fungi virulence. (rezk,2009)

Bioassay procedure: The virulence of the evaluated fungi was assayed (i) with the original isolates before subculturing on artificial media (ii) after subculturing on artificial media, and (iii) after in vivo passage in the insect.

      Adult individuals of the mealy bug, I. seychellarum were assayed on citrus trees (30 insects/ young tree) grown in a green house and the trees were sprayed with spore suspension (1.7X10⁸ spores /ml). Six treatments (sub cultures) were used. mother culture, 1^st, 2^nd, 3^rd, 4^th and 5^th passage culture through artificial medium and an insect host Icerya seychellarum (Hemiptera: Monophlebidae) beside the untreated check three replicates were considered one treatment. The fungi virulence was measured through daily calculation of mortality percent within mortality 14 days post fungal application. Thisprocedure was done according to (Asghar, 2013).

Statistical analysis: The percentages of corrected cumulative mortalities (Abbott, 1925) of each treatment and the mean were calculated with Microsoft excel program (means ± SD). And the data were analyzed with the Lpd-line program to calculate LT50 values (Finney, 1971).   

Results and Discussion

       The entomopathogenic fungi are known to lose their desirable features after repeated sub-culturing declining their virulence through successive passages (subcultures). Verticillium lecanii, which lose virulence, may sometimes be restored to their former potency by passing them through their insect host. The results of the effect on virulence of V.lecanii (Zimm.) Viegas passage through an artificial medium and an insect host I. seychellarum are recorded in Tables (1 and 2) from which, it could be deduced that. The mortality and LT50          values of the adult individuals of the mealy bug I. seychellarum exposed to mother and its obtained was according to differed the tested subcultures, time of exposure and the used medium at a single concentration of 1.7×10⁸ spores/ml. Daily recorded mortality percent proved that the lethal effects of the tested cultures were systematically arranged with increasing the time of exposure in all cases (subcultures) reaching to 94.4, 79.8, 51.7, 44.9 and 36% mortality percent in treatment with 1^st, 2^nd,3^rd,4^th and 5^th sub cultures, respectively when passaged through the MYB artificial medium compared with complete death (100% mortality ) of the insect population treated with the mother culture after 13 days. These results proved that the virulence of V. lecanii against I. seychellarum was decreased with increasing its passage's number through the MYB artificial medium. This was clear when the LT₅₀ values of the different passages through the artificial medium (MYB) were compared. The lowest value of LT₅₀ for the zero passage is 4.6 days followed by 5.8, 7.4, 11.3, 13.9 and 17.7 days for the five passages cultures, respectively (Table 2).

      Moreover, these results also confirmed that the passages of V. lecanii through a suitable insect-host, I. seychellarum have an increase of their virulence, showing the lowest value of LT₅₀  (4.6 days) for the zero passage followed by 5.3, 6.6, 8.4, 9.0 and 9.47 days for the five derived passages (subcultures), respectively . Comparing the five passages through the natural insect-host, I. seychellarum with the used artificial medium, it was found that virulence of subcultures obtained form the natural insect host was increased by 1.9 fold relative to that of subcultures obtained from the artificial medium Bo base on their LT₅₀ value.

     The obtained results are in agreement with Nagaich (1973) who found loss of virulence of V.lecanii (Zimm.) after two or three subculturing. Meanwhile, some strains need to be successively subcultured 10 to 12 times before a significant decline in virulence is occurred.   

      On the other hand, Asghar(2013) reported on the effects of repeated subculturing of Beauveria bassiana and Metarhizium anisopliae in vitro and their passages through insects and their virulence against Uvarovistia zebra. The virulence of both fungi was reduced after four subcultures in Potato Dextrose Agar media (PDA), but this reduction was not quite significant for B. bassiana. Attenuated fungi obtained from the fourth subculturing were passaged through 3^rd effects instar nymphs of U. zebra. The insect passage was repeated 2 times and virulence of the fungi was evaluated by its lethal. Following passage there was a small, but non-significant increase in the virulence of the fungi.

Table (1): Mortality effects on Icerya seychellarum at 1.7×10⁸ spores/ml of Verticillium lecanii derived from different passages, maintained on MYB medium and it's Insect-hos

Values are shown as mean ± SD

Table (2): LT₅₀ values on Icerya seychellarum exposed to Verticillium lecanii (Zimm.) derived from    different passages maintained through artificial media and an insect host ( I. seychellarum)