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Efficiency of Metarhizium spp. (Sorokin) Strains and Insecticides Against Cotton Mealybug Phenacoccus solenopsis (Tinsley).

Byline: Aziz Ahmed Ujjan, Muhammad Ali Khanzada, A.Q. Mahar and Saleem Shahzad

Abstract

We conducted a study to assess the insecticidal potential of the Hypocreales entomopathogenic fungi (epf) Metarhizium spp. strains, insecticides and their mixture for control of cotton mealybug (CMB) under laboratory, screen house and field conditions. The strain PDRL526 was the most effective amongst the strains of Metarhizium spp. at laboratory bioassays. It caused 50% mortality (LT50 value) of adult cotton mealybug at 5.2 days (LT50 value = median lethal time) after the application of 1.57x105 spore/cm2 (6.3x1012 spores/acre) inside the bioassay chamber. Therefore, the strain PDRL526 was selected to study at the screen house and field trials. The insecticide Lambda- Cyhalothrin was highly effective with 50% lethal concentration (LC50 value) (1.12 g/ml) followed by Acetamiprid (1.17 g/ml), Abamectin (1.62 g/ml), Imidacloprid (1.67 g/ml), Chlorpyrifos (2.09 g/ml) and Bifenthrin (3.05).

The insecticide Imidacloprid showed the best compatibility (95.2%) to the strain PDRL526; therefore, Imidacloprid was selected for screen house and field trials. The strain caused adult CMB mortality after (LT50) 13.8 and 19.6 days by using 6.3 x1012 spores/acre, under screen house and field conditions, respectively. The strain's application in combination with insecticide Imidacloprid (20 g a.i. /acre + 6.3x1012 spores/acre) showed a positive toxicity/virulence to CMB population at screen house and field trials with LT50 6.57 and 8.4 days, respectively. Along with the pest mortality, the yield of seed cotton/plant, increased with the spray of spores of the strain PDRL526, alone or in combination with Imidacloprid, at screen house and field trials as compare to control treatments. The study confirmed that M. anisopliae strain PDRL526 is effective against CMB.

Key words : Mycoinsecticides, entomopathogenic fungi, neonicotinoid insecticides, biological control.

INTRODUCTION

CMB, the cotton mealybug (Phenacoccus solenopsis Tinsley), had not posed any severe threat as crop pest until the end of 1990, when Watson and Chandler (2000) identified and reported its pest habit on several plants. Its pest habit was reported for the first time in Pakistan (South Asia) in the year 2005 and became a common (acclimatized) pest in Pakistan (Anonymous, 2008a) and India (Anonymous, 2008b; Nagrare et al., 2008). It caused heavy losses in cotton belts (Sindh and Punjab locations) in Pakistan (Anonymous, 2006, 2008c; Zaka et al., 2006; Kakakhel, 2007). CMB is difficult to control with low doses of chemical insecticides, because it has complex layer of wax that protects it against the contacts with pesticides. Therefore combinations between insecticides and biocontrol agents which can biodegradate this complex layer of wax (e.g. using specific enzymes) could present efficient results for CMB control (Fuchs et al., 1991).

The habit of CMB is sap sucking, which is also a defensive mode of nutrition against chemical contact pesticides (Fuchs et al., 1991), therefore the over doses are required to check insect pests. Since the hazards of insecticides and their residue in agro- products posed the concerns regarding human environment and health, the researchers look for organic farm or pesticide residue free products. Several remedies have been probed under biocontrol of diseases and pests of crops (Pimentel, 2009). Amongst the biocontrol agents, entomopathogenic fungi (epf) serve as mycoinsecticide (Faria and Wraight, 2007; Brand et al., 2012).

Members of the genus Metarhizium (Metschnikoff) Sorokin, also called green muscardine fungi, have been used against wheat chafer beetles Anisoplia austriaca and sugar beet curculio, Cleonus punctiventris (Metschnikoff, 1884; Lord, 2005). At present, the world has about 60 commercial mycoinsecticides based on Metarhizium spp. (Faria and Wraight, 2007); the most strains of M. anisopliae have a broad and divergent host range. M. anisopliae strains were reported effective against potato aphid (Myzus persicae), cabbage seed weevil (Centrorhynchus assimilis), cabbage flea beetle (Psylliodes chrysocephala), mustard beetle (Meligethes aeneus) (Butt et al., 1998) and mustard aphid (Lipaphis erysimi) (Ujjan and Shahzad, 2012). M. anisopliae was also used against black wine weevil (Otiorhynchus sulcatus) a pest of various ornamentals (Vestergaard et al., 1995).

Some studies reported the potential of epf against P. solenopsis (Makadia et al., 2009; Kumar et al., 2012; Banu et al., 2010). Although, there is appreciable amount of work carried out on other biocontrol agents, use of epf against CMB is unexplained in Pakistan. Therefore, the work was carried out to assess the insecticidal potential of epf strains for their assessment against CMB under laboratory, screen house and field conditions alone and with combination of the suitable insecticide formulation.

MATERIALS AND METHODS

Collection of samples

The team collected samples from Sindh, Pakistan. Live and dead insects with the symptoms of diseases were collected and processed for fungal isolation following the procedure of Goettel and Inglis (1997). The fungi were identified after Samson et al. (1988), Humber (2012), Barnett and Hunter (1988) and Domsch et al. (1980). The known isolates of epf were received from ARS Collection of Entomopathogenic fungal cultures, USDA-ARS RW Center for Agriculture and Health, USA. Every strain that was isolated from insect cadavers was allotted a collection number with respect to its host, collection time/date and area and stored at 20C after pure colony inoculation inside PDA poured slants.

Insect rearing

Insects were cultured on their host plants (cotton). Plant saplings grown in 15.24x20.32 cm earthen pots filled with loamy soil and humified cow dung (4:1 ratio). Each pot was kept at 14:10 h (light: dark) photoperiod. The seedlings were placed in a large chamber and covered with a fine net cloth supported by steel rod scaffoldings (76.2x17.7 cm) under natural light conditions of screen house. The pots were managed with cultural agronomy. The healthy adult insects were transferred to the saplings. The heavily infested plants were transferred to the laboratory.

Laboratory bioassay

The insect infestation on plant was washed with 0.01% sodium hypochlorite solution followed by sterilized distilled water. The required numbers of insects of respective life stages were transferred to bioassay chambers by using (zero size) camel hairbrush and nylon tip forceps. The entomopathogenic fungal spores were collected from 15 days old culture growing on PDA medium. The culture added with 5 ml sterile water and 0.02% Tween80 (v/v). The spores were slightly eroded with spatula and the spore containing solution transferred to a test tube. Hemocytometer used to count the epf spores per ml and the numbers of spores/ml were adjusted through dilution formula (Goettel and Inglis, 1997). The known number of insects per bioassay chamber (9 cm diameter or 63.6-cm2 area Petri plate) were infected with 1 ml of epf spores using an insulin syringes BDTM of 26 gauge needle. This micro spray technique adjudicated to consume 1ml spore suspension per bioassay chamber.

Each strain was applied to insects inside the bioassay chambers with 1x107 spores' concentrations (1.57x105 spore/cm2 or 6.3x1012 spores acre). Each treatment was replicated 5 times. Another set of treatments was assayed with water and 0.02% Tween80 solution as control. The insect population of the each treatment were noted every day for live and dead numbers. The percent mortality calculated through Abbott formula in comparison with control mortality (Abbott, 1925). Time dose mortality probit analysis was applied for the most probable lethal time of the insect. The epizootic symptoms of cadavers inside bioassay chambers were observed and analyzed by microscopic examination and inoculation of insect on PDA medium. After the confirmation of this preliminary test, the isolate was marked for further bioassays and experiments.

The known concentration of insecticides were assayed on the insect population inside the bioassay chamber and the number dead/alive insects were noted after24 h and the data was analysed for determination.

Compatibility test

For compatibility assessment of insecticides, the amount of water and pesticides' concentrations were used according to the recommended concentrations of the insecticide. The epf strain was examined for compatibility with chemical insecticides under the reference to Neves et al. (2001).

Screen house bioassay

The screen house assays were conducted at Pest and Disease Laboratory Green House facility, University of Karachi, Pakistan. The screen house was protected with a steel net of 2.5 cm2 and nylon net cover of 2 mm2 for protection from birds, insects and light. The large size (2 feet2) earthen pots were filled with sun dried loamy soil and cow dung manure (3:1 w/w). The pots were irrigated two days before sowing the seed. The variety of cotton (Variety NIAB 78) was selected based on their known susceptibility to test insect. The cottonseeds were soaked in water for 12 h before the sowing. Three young seedlings were kept intact and remaining plants were thinned after a foot height growths, remaining two were also thinned and one plant was maintained per pot. All the recommended fertilizers and irrigations were followed. Each plant was infested with 05 female insects, 15 days earlier to spray regimes. The spray regimes were started in June 2010.

The combined effect of epf and chemical pesticides were assessed by combined application of selected epf spore concentration with chemical pesticide, in the light of laboratory bioassay results of the fungal strains and pesticides. The best compatible pesticide was selected for synergistic application in screen house bioassays. Tween80 (0.02%) aqueous solution was mixed with epf spores grown on broken rice grains through single- phase fermentation (SPF). The numbers of harvested spores per gram of substrate are counted by using hemocytometer and the required concentration made over. Each treatment was sprayed with epf spores (6.30x1012 spores/acre); the epf spores concentration made in hand carry sprayers. Each sprayer was used against five replicates of single treatment. The control plants were sprayed with the spores and pesticide free Tween80 (0.02% v/v) solution.

The recommended doses of the most compatible insecticide (Imidacloprid) were used for CMB (20 g/acre). Population of insects on plant was counted before the spray and the changes in populations noted at different time intervals (01, 05, 10, 20 and 30 days) after the spray. The adult CMB population was counted on 10 cm length on twig and for instars; CMB leaf midrib area of test plant was counted. At the end of season, plants were harvested and their lint + seeds from dehiscent capsule (cotton) were collected and weighed. The differences between control and among treatments were analyzed. The experiment was completely randomized designed and the mortality percentage was corrected using Henderson and Tilton (1955) formula. The LT50 of the treatments were analyzed using probit analysis..

Field bioassay

The field applications were carried from April to September 2011. The test crop was cultivated in three plots with area of (20x10 feet) for each test. The plot-to-plot distance was about 12 feet. The soil and seedbed was prepared and seed sowed as per recommendations and according to agronomic protocol and procedures. After germination, 30 plants were maintained in each block with 2 feet plant-to-plant and 2.5 feet row-to- row distance. All plants were artificially infested with CMB five females per plant prior to 15 days of the spray treatments. Each plot was sprayed with the epf using low volume sprayer of 16L capacity, with prevention to cross contamination. The fungal spores and pesticide concentrations were applied in same amount as in screen house bioassay. Each plot was sprayed with insecticide (a.i. 20g/acre or 91.8 mg/plot) and epf spores (6.3x1010 spore/acre or 2.89x1012/plot) in combined or alone, preparations in aqueous dilution.

The spore treatments were also added with 0.02% Tween 80 as emulsifier. A plot was sprayed with the same volume of 0.02% Tween80 sterilized aqueous solution as control. The

Table I.- Details of different M. anisopliae isolates/strains with reference to their collection and virulence (LT50) to cotton mealybug (Phenacoccus solenopsis) adults at laboratory bioassays.

###Strain code###LT50 SE days###Source###Fungi###Region###Host###Habitat

###PDRL18###17.692.1###Local isolate###M. anisopliae###Karachi, Pakistan###Phenacoccus sp.###Cotton

###PDRL 116###13.51.3###Local isolate###M. anisopliae###Karachi, Pakistan###B. tabaci###Okra

###PDRL 129###22.403.2###Local isolate###M. anisopliae###Khairpur, Pakistan###B. tabaci###Cotton

###PDRL 137###15.552.9###Local isolate###M. anisopliae###Larkana, Pakistan###Rice stem borer adult###Paddy

###PDRL 174###16.997.4###Local isolate###Metarhizium sp.###Hyderabad, Pakistan###B. tabaci###Cotton

###PDRL 220###18.963.8###Local isolate###Metarhizium sp.###Khairpur, Pakistan###B. tabaci###Cotton

###PDRL 269###14.882.5###Local isolate###Metarhizium sp.###Khairpur, Pakistan###Phenacoccus sp.###Cotton

###PDRL 526###5.244.6###ARSEF (strain 1912)###M. anisopliae###Mexico###Homoptera###NA1

###PDRL 711###10.653.2###ARSEF (strain 3605)###M. anisopliae###N.A., Pakistan###Acrotylus sp.###NA

###PDRL 738###11.068.3###Local isolate###M. anisopliae###Larkana, Pakistan###Scirpophaga incertulas###Paddy

###PDRL 744###15.845.8###Local isolate###Metarhizium sp.###Khairpur, Pakistan###Chilo infuscatellus###Sugar cane

###PDRL 1043###15.616.3###ARSEF (strain 1729)###M. pingshaense###Tamil nidu, India###Nilaparvata lugens###Green house

Number of live adults and instars (CMB) population was randomly counted on 1 cm twigs (for CMB adults) of five plants in the plot before spray (day 1) and after 5, 10, 20 and 30 days of spray regimes. The differences of populations were calculated between control and treatments. Percent mortality was corrected using Henderson and Tilton formula (1955). The seed cotton yield of treated and control plants was also noted.

RESULTS AND DISCUSSION

Epf isolates

There were 12 Metarhizium spp. strains collected at laboratory culture collection, of which 09 were locally isolated and 03 received from abroad (Table I). The local isolates consisted of a huge number (1183) of non-target fungi i.e. Aspergillus sp., Cladosporium sp., Alternaria sp., Fusarium sp., Nigrospora sp., Dreschslera sp. and others, from insects. The isolation and assessment work emphasized over well-reported epf. The fungal contaminant issue reported as common hurdle (Goettel and Inglis, 1997).

Lab. bioassays

PDRL526 M. anisopliae caused higher virulence (LT50 5.24 days) than other 12 Metarhizium spp. strains (Table I). The strain was therefore selected for further quality and virulence tests in screen house and field conditions on CMB populations (in vivo) infested on cotton plants.

There are some reports available about the strains of M. anisopliae that were virulent to papaya mealybug (Paracoccus marginatus) and CMB in vitro conditions (Anonymous, 2010; Kumar et al., 2012; Banu et al., 2010; Nagrare et al., 2011). The report supports the present study. Other 11 strains of Metarhizium spp. were virulent with lower results against the CMB (Table I). This is due to their lower pathogenic attributes, as it is known factor that the different epf strains have different effects on same host insect, even if the strains are of the same species.

Insecticide toxicity and compatibility

Insecticide toxicity test was conducted to CMB using six insecticides of different groups for LC50 after 24 h (Table II). The insecticides were assessed for the selection of the best suitable insecticide for combine/synergistic application to epf at screen house and field bioassays. The insecticide lambda-cyhalothrin found highly toxic to CMB with LC50 1.12 ppm followed by acetamiprid (1.17 g/ml), abamectin (1.62 g/ml), imidacloprid (1.67 g/ml), chlorpyrifos (2.09 g/ml) and bifenthrin (3.05 g/ml) against CMB adult population inside bioassay chambers after 24 h of application (Table II).

Insecticide imidacloprid found more compatible to epf strains, when it examined for its effect on fungal colony and spore growth on culture media poised with its the recommended dose (Table III). It showed higher compatibility (95%) to the epf strain (Table III), therefore the insecticide preferred to be studied in screen house and field bioassays regimes.

Table II.- LC50 of pesticide concentrations at 24 h time interval, against cotton mealybug (P. solenopsis) adults in laboratory assays.

###95% confidence###Chi2

###LC50

Pesticide###limit###(dfa=48)

###(g/ml)

###Lower###Upper

Abamectin###1.624###1.454###1.829###366.6

Acetamiprid###1.170###0.761###1.585###4941.0

Bifenthrin###3.054###2.615###3.787###252.0

Chlorpyrifos###2.098###2.009###2.197###45.5

Imidacloprid###1.671###1.519###1.852###286.2

Lambda###1.127###.892###1.367###1771.8

Cyhalothrin

Table III.- Percent compatibility (T-value) of M. anisopliae strain PDRL526 with chemical insecticides.

###Insecticide (recommendation###PDRL526

###g/ml)

###Abamectin (10)###65.575.2b

###Acetamiprid (25)###86.812.1a

###Bifenthrin (25)###38.02.6d

Chlorpyrifos (400)###74.59.3b

Imidacloprid (200)###95.26.8a

Lambda Cyhalothrin (10)###55.71.2c

Control (00)###1000.1a

Ahsan (2007) reported that insecticide abamectin and lambda cyhalothrin showed LC50 0.68 and 1.2 g/ml against CMB adults that supports our results. No studies are available on the effect of imidacloprid, acetamiprid, bifenthrin and chlorpyrifos against P. solenopsis.

Imidacloprid was found highly compatible at 200 g/ml with epf strains in the present study.

There are several reports on compatibility of Imidacloprid with epf strains (Kim and Kim, 2007; Alizadeh et al., 2007; James and Elzen, 2001; Quientela and McCoy, 1998). The insecticides imidacloprid, therefore, holds promise for combined use in integrated pest management (IPM) strategies with epf.

Screen house bioassays

The strain sustained stress on CMB population with negotiable variation of virulence (LT50 13.8 days) under screen house conditions. The strain protected the plant which benefited and increased seed cotton yield after treatments (11.5 g/plant) as compared to control (7.0 g/plant) (Fig. 1). The synergistic effect of insecticide and epf strains geometrically increased the insect mortality and caused decrease in LT50 values (6 .5 days) under screen house conditions. The strain boosted cotton yield (11.5 g/plant) as compared to control treatments (7.0 g/plant), and the plants yielded double under synergistic effects of insecticide and epf (23 g/plant) (Fig. 1, Table IV).

The virulence of the strain reduced at screen house and consequent field trials (Table I, IV andV), Kumar et al. (2012) reported same observations that the virulence of epf strains decreased during in vivo bioassays against another group of insect pests, which supports the present study. The difference in virulence might be attributed to the higher abiotic and biotic stress like sunlight (UV radiation), wind, humidity deficits, symbiotic organisms at insect body and phyloplane and allelopathic compounds (Thomas and Jenkins, 1997; Hallsworth and Magan, 1994; Rangel et al., 2004; Inglis et al., 1997; Goettel and Inglis, 1997; St-Leger, 2008).

Table IV.- LT50 caused by M. anisopliae strain PDRL526 concentration and chemical pesticides 6.30x1012 spores + 20 g a.i. insecticide per acre) sprayed alone, in combinations with insecticide and insecticide alone against cotton mealybug (P. solenopsis) adults under screen house conditions.

###95% confidence###Chi2

###LT50

Strain###limit###(dfa=48)

###(days)

###Lower###Upper

PDRL526###13.812###10.888###17.852###253.5

Imidacloprid###13.132###5.319###67.010###979.9

PDRL526 +###6.571###3.569###10.355###600.7

Imidacloprid

It appears that the screen house application of the M. anisopliae for the management of CMB was carried out for the first time during our studies, since no report on screening of M. anisopliae under screen house conditions is available against (P. solenopsis) CMB. Although, the Metarhizium spp. is widely used as mycoinsecticides against variety of insect pests, even these are popular to control Hemiptera (Faria and Wraight, 2007).

The epf and insecticide synergism showed higher mortalities of insect populations than separate use of insecticide and epf. It suggests that combined use of imidacloprid with the M. anisopliae strain helps to start the fungal infection in environment by decreasing insect resistance in tri- trophic levels (Ambethgar, 2009; Roditakis et al., 2000; Quintela and McCoy, 1997, 1998; Anderson et al., 1989; Hassan and Charnley, 1989). The synergistic treatment promoted the higher yield of cotton seeds/plant. O'Brien (2009), Kan Kang et al. (1996), Ownley et al. (2010) and Hu and St-Leger (2002) reported that M. anisopliae has potential to support plant growth in addition to parasitize the insects. It appears that the present report on the combined application of entomopathogenic fungi and insecticide for the control of Phenacoccus solenopsis is very promising, since no prior report is available on synergistic use of entomopathogenic fungi and imidacloprid to CMB.

However, similar results were found with other fungal genus, with a combination of imidacloprid and Beauveria bassiana strain PDRL1187, that was suitable against mustard aphid (Lipaphis erysimi) under field condition (Ujjan and Shahzad, 2014).

Table V.- LT50 value of M. anisopliae strain PDRL526 applied on cotton mealybug (P. solenopsis) adults with single application and in combination of imidacloprid 20 g + 6.30x1012 spores per acre or single dose of imidacloprid concentration under field conditions.

###95% Confidence###Chi2

Strain

###LT50

###Limit###(dfa=48)

###(days)

###Lower###Upper

PDRL526###19.610###15.779###25.803###136.2(22)

Imidacloprid###14.735###10.058###24.280###296.1(23)

PDRL526 +###8.447###6.432###10.870###188.2(23)

Imidacloprid

Field bioassays

The field application of epf showed a comparative decrease in virulence (LT50 19.6 days) as compare to less stressed conditions of screen house (Table V). Although the cotton yield and insect mortality data suggested the strain was efficient at field. When the insecticide applied with the combination of the fungal spores the mortality synergized i.e. decreased LT50 value (8.4 days) and seed cotton yield (14.5 g/plant) increased (Table V, Fig. 2). The visual evidences of died insects also confirmed the mortality of the insect population in field, when the cadavers incubated on mycological medium (PDA), the M. anisopliae growth assured the hypothesis about the virulence of the strain (Fig.3). While the strain efficiency was multiplied to check the CMB population and seed cotton yield, when applied with the insecticide.

The single application of the strain PDRL526 at field condition showed a bit reduction in virulence as compared to the screen house condition. The same results were also reported by Nagrare et al. (2011). However, the strains showed protective virulence in field (Table V). The imidacloprid 20 g a.i. per acre sustained virulence and caused insect mortality 50 to 60% after 30 days of application, same higher toxicity is reported by Lysandrou et al. (2012), when they used imidacloprid @ 125 g a.i. per hectare which caused 100% mortality. Suresh et al. (2010) reported, imidacloprid 20 g a.i. per acre wiped out the CMB population after 3 days of application. Dhawan et al. (2009) reported imidacloprid 200SL @ 900 ml per hectare (aprx. 72 g per acre) reduced 81% CMB population at field conditions. Tanwar et al. (2007) recommended imidacloprid @ 20 g per acre. The combined application of imidacloprid and epf strains showed synergism against CMB populations.

The strain under present study increased plant yield in cottonseed under field conditions as compared to control plants (Fig. 2). The strain in combined applications with imidacloprid insecticide synergized in insect mortality as well as in cottonseed production (Fig. 2, Table V). Some strains of M. anisopliae reported to promote plant growth in addition to insect control (O'Brien, 2009; Kan Kang et al., 1996; Ownley et al., 2010; Hu and St-Leger, 2002), which suggests the PDRL526 strain has new dimensions for these studies.

It is, therefore suggested that the strain PDRL526 M. anisopliae has sustainable mycoinsecticidal attributes, and it can be utilized against cotton mealybug.

ACKNOWLEDGEMENTS

The exotic strains used during the present studies were kindly provided by ARS Collection of Entomopathogenic fungal cultures, USDA-ARS RW Center for Agriculture and Health, USA, that is gratefully acknowledged.

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Author:Ujjan, Aziz Ahmed; Khanzada, Muhammad Ali; Mahar, A.Q.; Shahzad, Saleem
Publication:Pakistan Journal of Zoology
Article Type:Report
Geographic Code:9PAKI
Date:Apr 30, 2015
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