Virulence of Five Isolates of Indigenous Beauveria bassiana Against Eggs and Nymphs of Bemisiatabaci Gennadius (Hemiptera: Aleyrodidae)

This research aims to study the virulence of five isolates of Beauveria bassiana to eggs and nymphs of Bemisiatabacion tomato. In the experiment eggs and second instar nymphs of B. Tabaci were used. Five isolates of the fungus, i.e.,WS, TD312, PD114, PA221, PB211,were tested. Conidial concentration of B. bassiana used were 108 conidia/ml. Experimental parameters included mortality of eggs and nymph and percentage of adult emergence. The results showed that all B. bassiana isolates tested were able to kill B. Tabaci eggs but with very low mortality (2-19%). Mortality of second instar B. Tabaci nymphs was dependent on the fungal isolates. Isolate WS had the highest virulence, which caused 70% mortality of 2nd instar nymphs, with aLT50 of 4.87 days. Nymphs of B. Tabaci were highly susceptible to B. bassiana infection compared with eggs. B. bassiana applicated to nymphs of B. Tabaci can decrease the percentage of adult emergence. CONTACT Trizelia trizelia@yahoo.com Department of Plant Pests and Disease, Faculty of Agriculure, Andalas University Kampuslimau Manis, Padang, West Sumatera, Indonesia. © 2021 The Author(s). Published by Enviro Research Publishers. This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY). Doi: dx.doi.org/10.12944/CARJ.9.1.07 Article History Received: 18 March 2020 Accepted: 22 May 2021


Introduction
Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is one of the main pests in tomato plants. 1 B. tabaci is a polyphagous insect that has many host plants such as ornamental plants, vegetables, fruits, and wild plants (weeds). 2 This insect make damage directly on plants by sucking the liquid, causing a physiological disturbance on plants, chlorosis on leaves, and disturb ripening of tomatoes. 3,4 In addition to direct damages, the pest cause indirect damage by the accumulation of honey dew produced by B. tabaci which leads to mold growth on foliage, and as a vector of Tomato yellow leaf curl virus (TYLCV) and more than 100 other begomo viruses. 5,6 The loss of yields due to B. tabaci attacks and yellow virus ranges from 20 to100%. 7 This pest was first found in Indonesia in 1938 on a tobacco plant. 8 The reproduction and spread of these pests are very fast, in fact, in a year, is able to produc till 15 generations. 9 Traditionally, synthetic insecticides have been used for controlling B. tabaci, but excessive and irrational use of insecticides has led to adverse effects on the environment. To reduce the use of pesticides, it is important to developing alternative safety control methods such as the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. B. bassiana is considered the most effective alternative control method of B. tabaci. It is is an entomopathogenic fungus with a wide host range, able to infect various kinds of insects, from pre-adult to adult. 10,11 B. bassiana infects insect by digestion, respiration, and particularly through the integuments of insect. 12 Beauveria bassiana have been used as a biological agent to control several insect pests. In laboratorium condition, B.bassiana was able to kill Crocidolomiapavonanalarvae till 80%. The mortality of larvae depends on isolates. 13 B.bassiana can kill several kinds of vegetable pests such as Spodopteraexigua, 14 S.litura, 15 Nezaraviridula, 16 and Eurydemapulchrum. 17 The dose of B. bassiana had a significant effect on adult of Aphis crassivora and B.tabaci. An increase of B. bassiana concentration markedly decreased adult longevity, period of reproduction, and fecundity of the two insects. 18 One of the essential criteria in selecting entomopathogenic fungi for commercial development is to have high virulenceto target insects. In order to evaluate the importance of virulent strains for an efficient biological control of B. tabaci on tomato, we analyzed under laboratory conditions the effect of five fungal isolates of indigenous B. bassiana on eggs andnymphs of B. tabaci.

Bioassay of B. Bassiana against B. Tabaci Eggs
The tomato leaf contained 20 eggs selected and treated with the fungus. Eggs were sprayed by conidial suspension of B. bassiana, then eggs were put into Petri dish on a moist filter paper. The assays was repeated five times. As control, the same number of eggs were treated with distilled water. The eggs were reared till their hatching. Eggs mortality and infection of first instar nymphs were assessed and recorded daily for eight days.

Bioassay of B. Bassiana against B. Tabaci Nymphs
The second instar nymphs of B.tabaci were moved on 30 days old tomato plants using a soft brush. Furthermore, 2 ml of the B. bassiana fungal suspensions, for each unit test, were sprayed on the insects using hand sprayer. For the control, theinsects were sprayed with distillate water. The tomato plants were put into a cylindrical tubularshaped plastic mica cage (high 60 cm, diameter 45 cm) and covered with gauze. The treatments were repeated five times, and every unit of treatment consisted of 10 nymphs. The mortality of nymphs was observed every day by counting the numbers of test insects that died until seven days after application (DAA).

Data Analysis
The data obtained were analyzed by ANOVA and followed by a test of Duncan's New Multiple Range Test (DNMRT) a significant level of 5%. Based on macroscopic observations, the fungal mycelial covered the eggs. Fungal mycelia on the surface of the eggs were seen four days after the inoculation of the fungus. Al-Deghairi 19 also reported that infection symptoms on the eggs of B. tabaci were observed on the 3 rd day of inoculation. Within four days from the treatment, the eggs that became subsequently infected by the fungal had little color change but appeared slightly shrunk when observed under the microscope. One week after treatment, most of the unhatched eggs became conspicuously shrunk and had fewer fungal out growths on the surface.
The results showed that conidia remained active and can infectnymphs successfully emerging from eggs. Occurrence mortality of 1 st instarnymphs is thought due to contact nymphs emerging from eggs with the conidia attached to the surface of the eggshell and on the leaves. Other researchers 23 also reported that B. tabaci eggs had low susceptibility to B.bassiana, with >91% nymphs successfully emerging from eggs. However, there is significant mortality of 1 st and 2 nd instar nymphs originating from the treated eggs. These results indicated that newly hatched nymphs probably acquired conidia from the eggs soon after hatching or from the leaf surface as secondary exposure. Trialeurodes vaporariorum eggs treated with entomopathogenic fungus Aschersonia aleyrodis did not become infected, but larvae that hatched from these eggs were infected. 24

Mortality of Bemisia Tabaci Nymphs
The results showed that B. bassiana isolate had a significant effect on the mortality of 2 nd instar nymph of B.tabaci. WS isolates were the most virulent isolates with the highest mortality namely 70.00% after seven days after the fungal application. PA221 isolate had a low virulence with a mortality of 36.0% ( Table 3).
The difference in the ability of B. bassiana isolates in killing B. tabaci nymphs is thought to be due to differences in viability of conidia or the ability to produce enzymes and toxins. The difference in mortality of C. pavonana larvae after B. bassiana application was caused by differences in physiological and genetic characteristics of the isolates. 25 Several researchers 26,27,28 stated that the germination of conidia and the ability to produce enzymes and mycotoxins during the infection process in insects affects insect mortality by B. bassiana.
This research showed that the mortality of nymphs of B. tabaci after application B.bassiana was influenced by isolate source. B.bassiana isolated from insects belonging tothe same taxon of the test insect (WS) was more virulent than that isolated from the plants. Based on the observed values, surveys of B. bassiana isolates collected from homopterans seems to be a suitable approach for silver leaf whitefly microbial control programs. Other  Besides beingable to infect the eggs,nymphal mortality also occurs in 1 st nymphs upon hatching from eggs contaminated by B. bassiana ( Table 2). The nymph mortality varied between isolates (F = 3.55; db = 5, 24; P> 0.0152). WS isolates produced the highest nymph mortality, namely 7.43%, while PA221 isolates only produced nymph mortality of 2.05%. In control, there was no mortality in nymphs.
researchers also reported that mortality of insect was dependent on the fungal isolates. Isolates or strains of entomopathogenic fungi isolated from the same taxon of the whitefly (Order Hemiptera, suborder Homoptera) were more virulent than the isolates from Lepidoptera, Coleoptera, and Hymenoptera. 23,29,30,31 There was a difference in LT 50 between isolates ( death, and most conidia were recorded on legs, wings, and thoraces of some adult cadavers. 32 Cherguiet al. 33 noted that dead individuals of Ceratitiscapitata were covered with a white mycelium characteristic of the fungus B. bassiana.

Adults Emergence
The results showed that B. bassiana applied to 2 nd instar nymphs of B. tabaci had a significant effect on the emerged numbers of B. tabaci adults.
Percentages of emerged adults under five fungal isolates can be seen in Table 4.
The application of fungus B. bassiana to the 2 nd instar nymph showed a significant effect on adult emergence. In control, the percentage of adult emergence was the highest at 98%, while in the treatment of WS isolates only 22% of nymphs became adult. The low rate of adult emergence was because a lot of some nymphs was killed before becoming an adult. B. bassiana applied to C.capitata larvae decreased the percentage of pupae formed

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.