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An improved cage system for the bioassay of Metarhizium anisopliae on Thrips tabaci (Thysanoptera: Thripidae)

An improved cage system for the bioassay of Metarhizium anisopliae on Thrips tabaci (Thysanoptera: Thripidae)
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   PLEASE SCROLL DOWN FOR ARTICLE This article was downloaded by: [Talaei-Hassanloui, Reza]  On: 28 September 2008  Access details: Access Details: [subscription number 903095330]  Publisher Taylor & Francis  Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK Biocontrol Science and Technology Publication details, including instructions for authors and subscription information: An improved cage system for the bioassay of Metarhizium anisopliae on Thripstabaci Thysanoptera: Thripidae) Hamid-Reza Pourian a ; Reyhaneh Ezzati-Tabrizi a ; Reza Talaei-Hassanloui aa  Department of Plant Protection, Campus of Agriculture and Natural Resources, University of Tehran, Karaj,IranFirst Published on: 08 August 2008 To cite this Article  Pourian, Hamid-Reza, Ezzati-Tabrizi, Reyhaneh and Talaei-Hassanloui, Reza(2008)'An improved cage system forthe bioassay of Metarhizium anisopliae on Thrips tabaci (Thysanoptera: Thripidae)',Biocontrol Science and Technology,18:7,745 —752 To link to this Article: DOI: 10.1080/09583150802290618 URL: Full terms and conditions of use: article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.  SHORT COMMUNICATIONAn improved cage system for the bioassay of   Metarhizium anisopliae  on Thrips tabaci   (Thysanoptera: Thripidae) Hamid-Reza Pourian, Reyhaneh Ezzati-Tabrizi and Reza Talaei-Hassanloui* Department of Plant Protection, Campus of Agriculture and Natural Resources, University of Tehran,Karaj, Iran(Received 28 March 2008; returned 29 April 2008; accepted 20 June 2008) The ability of thrips and other minute insects to escape from conventional assay cups ledto development of a successful assay chamber. In the present study, we evaluated threebioassay systems for thrips; Tashiro cage, microtube assay and leaf sandwich assay andintroduced an improved thrips entomopathogenic bioassay system (TEBS). Ourobjective was to investigate the effect of   Metarhizium anisopliae  on  Thrips tabaci   usingan easy-to-use and inexpensive holding chamber that would produce bioassay resultswith acceptable levels of accuracy and precision. In our assays, we found that escape of second-instar larvae from TEBS was significantly less than those of other methodswhich we tested. Our results indicated that our assay system was more suitable than theother conventional assays for the bioassay of entomopathogenic fungi on  T. tabaci  . Keywords:  Thrips tabaci  ;  Metarhizium anisopliae ; bioassay chamber; entomopathogenicfungus; escape Onion thrips,  Thrips tabaci   Lind (Thysanoptera., Thripidae) are distributed worldwide andare a major pest of greenhouse crops. In most target crops, use of synthetic pesticides is themost commonly used option for controlling thrips. Their ability to rapidly developresistance to synthetic chemical insecticides (Brodsgaard 1994; Reuda and Shelton 2003;MacIntyre-Allen, Scott-Dupree, Tolman, and Horris 2005) has stimulated research onalternative methods of control.Entomopathogenic fungi are currently being investigated for the control of manyimportant insect pests on various crops around the world and some are commerciallyavailable (Thungrabeab, Blaeser, and Sengonca 2006). Carl (1975) reported that  Neozygites parvispora  has been found frequentlyon  T. tabaci   but under field conditions, the funguswasless successful than in the greenhouse. Gillespie (1986) and Fransen (1990) reported that T. tabaci   was susceptible to  Metarhizium anisopliae ,  Beauveria bassiana ,  Paecilomyces fumosoroseus  and  Verticillium lecanii   in laboratory studies .  Application of   B. bassiana  and M. anisopliae  has been reported to reduce thrip populations in greenhouse crops such ascucumber, chrysanthemum, roses and carnations (Bradley et al. 1998; Murphy, Morisawa,Newman, Tjosvold, and Parella 1998; Jacobson, Chandler, Fenlon, and Russell 2001;Azaizeh, Gindin, Said, and Barash 2002; Ludwig and Oetting 2002; Shipp, Zhang, Hunt,and Ferguson 2002; Ansari, Shah, Whittaker, Prasad, and Butt 2007). * Corresponding author. Email: published online 8 August 2008 Biocontrol Science and Technology ,Vol. 18, No. 7, 2008, 745    752 ISSN 0958-3157 print/ISSN 1360-0478 online # 2008 Taylor & FrancisDOI: 10.1080/09583150802290618  D o w nl o ad ed  B y : [ T al a ei - H a s s a nl o ui ,  R e z a]  A t : 05 :47 28  S e p t e mb e r 2008  The search for new isolates requires a reliable bioassay system. Because of larvaeescaping from bioassay chambers and its negative effect on the results of experiments, wehave to use a chamber which is escape-proof and keeps leaf disks fresh for an extendedperiod. Reuda and Shelton (2003) developed a common bioassay method, referred to asthrips insecticides bioassay system (TIBS) for evaluating the effect of insecticides. Thismethod is not suitable for the bioassay of entomopathogenic fungi because it does not keepleaf disks fresh enough for the increased monitoring period required for entomopathogenicfungi (unpublished data).The ability of thrips to escape from conventional assay systems such as the Tashiro cage(Tashiro 1967), microtube assay (Abe and Ikegami 2005) and leaf sandwich assay (Ugine,Wraight, Brownbridge, and Sanderson 2005) led us to develop an improved system whichwe call the thrips entomopathogenic bioassay system (TEBS). We tested this system tobioassay  M. anisopliae  against the onion thrips,  Thrips tabaci   and compared the results of this method with the other above-mentioned common systems for larval escape andmortality. We describe this system as an efficient, easy-to-use and economical holdingchamber with acceptable levels of accuracy and precision.A colony of   T. tabaci   was established from greenhouse-collected specimens oncucumber plants in Karaj, Iran and reared on cucumber ( Cucumeris sativus  Var.  sultan )leaves in plastic dishes at 25 9 1 8 C, 60 9 5% RH and 16:8 h (L:D). These ventilatedpolygonal hyaline dishes, 11 cm in diameter, were filled with a 1-cm thick layer of 1% wateragar. Freshly excised cucumber leaf disk, 8 cm in diameter, were placed upside down ontothe water agar and 25    30  T. tabaci   females from the stock culture were placed on the leaf disk for 12 h for egg laying. Thereafter, females were transferred to new dishes. The eggsobtained in the old cages were reared until the thrips reached the age desired for theexperiments. On the fourth day, a 9-cm 2 piece of cardboard was placed on the leaf diskallowing second-instar larvae to pupate readily in the seams of cardboard and reducing thenumber of escapes, which occur if the larvae are not able to find suitable habitats forpupation.An isolate of   M. anisopliae  EUT118 ( Cydia pomonella  origin) was cultured onSabouraud’s dextrose agar with 1% yeast extract (SDAY) and incubated at 25 9 1 8 C, 60 9 5% RH and 16:8 h (L:D). Conidia were harvested from up to 16-day-old cultures andspores were suspended in 20 mL sterile distilled water containing 0.02% Tween 80. Theconcentration of conidial suspension was estimated with a haemocytometer. Six serialconcentrations, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 conidia/mL were used. The viability of conidia was determined by spreading 0.1 mL of conidial suspension (10 5 conidia/mL) onthree PDA plates (5 cm in diameter). These plates were incubated at 25 9 1 8 C. Thepercentage germination was examined after 20 h by counting 100 conidia on each plate.The viability of conidia was recorded as more than 95% during the period of experiments.The bioassay chamber consisted of the common hyaline canisters used for storage of 35 mm photography film, 5 cm in the height and 3 cm in diameter. We reduced the heightto 3 cm by cutting off the top 2 cm and made a hole, 1 cm in diameter in the lid andcovered it with 200 mesh polyester netting. These small chambers were filled with a 0.5-cmthick layer of 1% water agar and cucumber leaf disks, 5 cm 2 were placed upside down ontothe water agar (Figure 1). Using this method, the freshness of the cucumber leaf disk lastedfor at least 3 days. Fifteen early second-instar onion thrips were then transferred onto theleaf disks in each canister. The chambers were placed in a refrigerator (5    7 8 C) for 15 min todecrease larval movement. Then 1 mL of each concentration of   M. anisopliae  EUT118conidia was applied to the early second-instars in the bioassay chambers using a potter746  H.-R. Pourian  et al.  D o w nl o ad ed  B y : [ T al a ei - H a s s a nl o ui ,  R e z a]  A t : 05 :47 28  S e p t e mb e r 2008  spray tower (Burkard, UK) with a nozzle size of 0.25 mm diameter and 0.7 kg/cm 2 pressure.Soon after fungal application, we counted the number of larvae for any escape. Theedge of each canister was covered with a layer of Parafilm † (Laboratory Film, Chicago,IL) and then lids of the film canisters were placed and sealed with additional Parafilm.These chambers were incubated at 25 9 1 8 C, 60 9 5% RH and 16:8 h (L:D). After 24 h,thrips escape and mortality were recorded daily for 7 days. Each conidial concentrationwas applied in four replicates with each replicate containing 15 larvae. Control larvae weretreated with distilled water (0.02% Tween 80). Bioassays were also conducted using theTashiro cage (Tashiro 1967), microtube assay (Abe and Ikigami 2005) and leaf sandwichassay (Ugine et al .  2005). Seven-day data of cumulative mortality were corrected forcontrol mortality by using Schneider Orelli’s formula (Pu¨ntener 1981), transformed andanalysed using the General Linear Model (SYSTAT 2000). Similar analyses were carriedout for the 7-day escape data for all the caging methods tested. The Tukey HSD test wasapplied for mean comparisons among treatments. The whole experiment was repeatedthree times and conducted on the basis of a completely randomised design (CRD).There were significant differences among the assayed systems for escape of second-instar larvae, considering six conidial concentrations and control ( F  3, 108  163.795,  P  B 0.001). Recorded mean escape percentages after application of 10 8 conidia/ml (5.8    6.2   10 3 conidia/mm 2 ) were 63, 53, 26 and 1.7% for Tashiro cage, microtube assay, leaf sandwich assay and TEBS, respectively. Mean comparisons of escapes grouped these fourassay-systems into the four different statistical groups (Tukey HSD,  P  B 0.01, Figure 2).Analysis of corrected 7-day total mortality data at six conidial concentrations and controlshowed that there were significant differences among assay systems ( F  3, 108  3.09,  P  B 0.01). Recorded mean mortality rates after treatment with 10 8 conidia/ml were 70, 75, 57and 91% for Tashiro cage, microtube assay, sandwich leaf assay and TEBS, respectively(Figure 3). Recorded mean escape and mortality percentages for Tashiro cage, microtubeassay, leaf sandwich assay and TEBS are presented in Table 1. In addition to significantdifferences among treatments for escapes and total mortalities, we observed greatdifferences in standard error (S.E.) values for mortality data regarding the first three Figure 1. Schematic and real design of TEBS. Some changes were made to the photography filmcanisters. These small chambers were filled with 0.5-cm thick layer of 1% water agar and cucumberleaf disk, 5 cm 2 was placed upside down onto the water agar. Biocontrol Science and Technology  747  D o w nl o ad ed  B y : [ T al a ei - H a s s a nl o ui ,  R e z a]  A t : 05 :47 28  S e p t e mb e r 2008  assay-systems and thought that variation of values for this parameter could be a goodcriterion for judgment of their accuracy and precision, and for comparing these assay-systems with TEBS. The results of   F  -test showed that there are significant differencesamong assay systems for S.E. values of thrips mortality ( F  3, 92  8343.4,  P  B 0.001). Theseassay-systems were placed into four different statistical groups (Tukey HSD,  P  B 0.05) withthe lowest value for TEBS (Table 2).A wide range of bioassay chambers has been used by various workers for disparateinsect species. For assays with small insects, it is often possible to use single leaf petioles orexcised leaves in small bioassay chambers (Butt and Goettel 2000). One problem which ismore important with entomopathogenic fungi in comparison with chemical insecticides isto delay leaf senescence as long as possible, because the effect of fungi on the host is slowerthan that of insecticides and therefore the monitoring period is longer. To solve thisproblem, various workers have used different methods or substrates. Our results indicatedthat TEBS is more suitable for the bioassay of onion thrips in comparison with the othermethods for having the lowest larval escape. With regard to the importance of food, wespeculated that the quality of food source could be an effective factor in retention of larvaewithin bioassay chambers. In the Tashiro cage, leaf disks are placed between two Plexiglaslayers and consequently leaves rapidly lose their freshness and quality due to the pressureon leaves and absence of oxygen on the first day after inoculation. In the microtube assay,due to the tiny and closed patch, the food source is paled and fades rapidly and sometimesthe plant sprout is infected with saprophytic fungi. Also in this way, handling a delicatetube system to transfer the larvae may cause the mesh of the microtube tip to be readily Figure 2. Mean percentage escape ( 9 S.E.) of second-instar larvae of onion thrips, recorded byusing four different bioassay chambers for the bioassay of   Metarhizium anisopliae  EUT118 on  Thripstabaci   at six conidial concentrations after 7 days. Values given with different letters are significantlydifferent from each other (Tukey HSD,  P  B 0.01). 748  H.-R. Pourian  et al.  D o w nl o ad ed  B y : [ T al a ei - H a s s a nl o ui ,  R e z a]  A t : 05 :47 28  S e p t e mb e r 2008
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