International Journal of Pest Management

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Reddy, Narasimha; Andhar University Khan, Akbar Ali; Andhra University, Botany Devi, Uma; Andhra University, Botany Sharma, Hari; ICRISAT, Biotechnology Reineke, Annette; Geisenheim Research Center, Phytomedicine

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Complete List of Authors:

International Journal of Pest Management

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Date Submitted by the Author:

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Treatment of millet (Sorghum bicolor) with the entomopathogenic fungus Beauveria bassiana to reduce infestation by the stem borer, Chilo partellus

Sorghum , entomopathogenic fungus, Beauveria bassiana, endophytism, Chilo partellus

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Keywords:

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Treatment of millet (Sorghum bicolor) with the entomopathogenic fungus Beauveria bassiana to reduce infestation by the stem borer, Chilo partellus Narasimha Reddy P. a, Akbar Ali Khan P. a, Uma Devi K.a*, Hari Sharma C. b, &

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Annette Reineke c aDepartment

of Botany, Andhra University, Visakhapatnam, 530 003, AP, India,

bEntomology

Division, ICRISAT, Patancheru, 502 324, AP, India, and

cForschungsanstalt

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Geisenheim, Fachgebiet Phytomedizin, Chemical Ecology,

Von- Lade-Str. 1, D-65366 Geisenheim, Germany

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Short title: Endophytic B. bassiana in sorghum to combat stem borer

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Abstract Experiments were conducted to test whether Beauveria bassiana (Balsamo) Vuillemin can become established endophytically in sorghum and thereby confer protection against the stem borer Chilo partellus. Four-week-old sorghum seedlings were treated with B. bassiana and the plants examined for the

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endophytic presence of B. bassiana, 30 and 60 d after treatment. Stem cultures derived from treated plants showed growth of B. bassiana. PCR amplification with fungal specific primers for a conserved region of the beta tubulin gene

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yielded a 360 bp product from DNA of B. bassiana isolate ITCC 4688 used for treatment of sorghum and sorghum plants treated with B. bassiana. The PCR

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products from the two were identical in sequence. In a subsequent experiment,

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sorghum plants treated and untreated (control) with B. bassiana were artificially infested 15 d post-treatment with stem borer larvae, and the extent of damage

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(stem tunneling and dead-hearts) by the stem borer compared. Forty percent of

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the control plants showed dead-heart while no plants in the B. bassiana-treated

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plot showed such development. In the surviving control plants, stem tunneling by shoot borer was 40% as compared to ~4% in B. bassiana-treated sorghum

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plants.

Key words: Sorghum; entomopathogenic fungus; Beauveria bassiana; endophytism; Chilo partellus

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*

Corresponding author: Uma Devi. K, Department of Botany, Andhra

University, Visakhapatnam, 530 003, Andhra Pradesh, India, Tel: +91-8912525582 Fax: +91-891-2755547, E-mail address: [email protected]

1. Introduction

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Sorghum bicolor (L.) Moench, a drought-tolerant, nutrient-rich millet, is extensively cultivated both for grain and cattle fodder in poor soils in rain-fed fields of the semi-arid tropics of India and Africa. Of the several insect pests

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infesting this crop, the stem borer Chilo partellus Swinhoe is the most serious, causing 18-53% yield loss (Gethi et al. 2001). Stem borer-resistant sorghum

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varieties are currently not available. Chemical methods used to manage stem

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borer are not only ineffective but also unaffordable to the resource-poor dry-land farmer.

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Beauveria bassiana, an entomopathogenic fungus with no known

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phytopathogenic activity, is able to become established endophytically in corn

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and confer protection against the stem borer Ostrinia nubilalis Hübner (Arnold and Lewis 2005; Wagner and Lewis 2000). B. bassiana has also been reported to be

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able to become established endophytically in plants of different habit: herbs, shrubs and small trees. It has been reported in potatoes, jimsonweed, cotton and cocklebur (Jones 1994), tomatoes (Leckie 2002), opium poppy (Quesada-Moraga et al. 2006), Theobroma cacao L. (Posada and Vega 2005), date palm (Gomez et al.

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2006), coffee (Posada and Vega 2006; Posada et al. 2007) and the bark of Carpinus caroliniana Walt. (Bills and Polishook1991). B. bassiana has previously been found to be pathogenic to sorghum stem borer in laboratory bioassays and field treatments (Maniania 1993; Uma Devi et al. 2001). The aim of this study was to determine whether B. bassiana can become

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established endophytically in sorghum tissues, through artificial inoculation, and thereby confer protection against stem borer. 2. Materials and methods

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B. bassiana isolate ITCC 4688 (Indian type culture collection, IARI, Delhi, India) that caused ~80% mortality of larvae of C. partellus in laboratory bioassays (Uma

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Devi et al. 2001) was selected for the experiment. It was mass-cultured on rice as

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described by Jenkins et al. (1998). The conidiating rice culture was directly used in some treatments, while for spray applications, the conidia on the rice culture

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were harvested (through sieving) to make an aqueous conidial suspension. A

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sorghum variety, ‘Annapurna’, which is extensively cultivated in south India

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and susceptible to shoot borer, was treated. A second generation larval population of laboratory-bred C. partellus derived from field-collected insects

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was used in the experiments.

The experiments were conducted twice during the Rabi season (January–April with temperatures between 27–32 ºC (day)/ 20–26 ºC (night)) in two consecutive years (2005, 2006). In the first experiment, sorghum plants were treated with B. bassiana to test whether the fungus could become established in the plant. When

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it was ascertained that the fungus could do so with no unfavourable effect on the plant’s growth or yield, the experiment was done a second time. In the second experiment, sorghum plants were artificially infested with C. partellus larvae post-treatment with B. bassiana, to test if the endophytic B. bassiana deterred damage of the plant by the borer.

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2.1. Experimental design

For experiments in the first season, we used a single plot with three replicates

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for each treatment with different treatments arranged as split plots (Lewis et al. 2001). Each replicate treatment block consisted of 100 plants totaling to 300 plants

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per treatment. The control plants (B. bassiana treatment) were grown in a plot 200

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m away from the treated plot with a rice plot in between, to prevent crosscontamination by B. bassiana conidia due to carriage by wind. The plants were

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grown in rows with spacing of 0.5 m between rows and 1 m between plants. For

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the second experiment, six 25 m x 25 m (0.25 ha) plots, each separated by at least,

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100 m were used, each plot being assigned to a different treatment. The betweenrow and between-plant spacing’s were similar to the first experiment. Usual

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agronomic practices of fertilizer application and manual weeding were done both in the treated and control plots. The field was irrigated prior to B. bassiana treatment. This was to maintain high humidity (favourable for fungal establishment) in the micro-environment of the plots. B. bassiana treatment was

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done in the evening when temperatures were considerably lower (25–28 ºC) than day temperatures.

2.2. Treatment with B. bassiana Four-week-old seedlings of sorghum were treated with B. bassiana. The mean

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height (base of the stem to the point of whorl) of the plants at this stage was ~18 cm. The plants were treated in two ways – aqueous conidial spray or with conidiating fungal culture on rice substrate.

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2.2.1. Treatment with aqueous conidial spray. Each plant was sprayed with ~10 ml of

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an aqueous conidial suspension (5 x 109 conidia ml−1) with 1% Tinopal LPW and

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0.001% Tween 80. The controls were treated with an equal volume of water with 1% Tinopal LPW and 0.001% Tween 80. Tinopal was used to protect conidia from

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damage caused by UV rays in sunlight (Inglis et al. 1995).

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2.2.2. Treatment with conidiating fungal culture on rice substrate. The 14 d-old conidiating culture of B. bassiana on rice (1012 conidia g) was placed in the leaf

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whorl (5g per plant). The controls were treated with rice (5g per plant) processed as for mass-culture of B. bassiana but with no fungus.

2.3. Assessment of growth and yield in B. bassiana treated sorghum

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The growth (plant height) and yield (grain weight) of B. bassiana-treated and control sorghum plants were compared to check whether B. bassiana had adverse effect on the plants. The plant height was measured twice – 30 d and 60 d after treatment. Twenty plants from each replicate were measured for height. The height was measured on the 30th day after treatment from the base of the stem to

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the tip of the top most leaf on the main tiller. On the 60th day, the height was measured from the base of the stem to the tip of the panicle on the main tiller. To assess yield, mature panicles from 20 plants from each treatment block were

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collected. The grain was threshed and its weight was measured.

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2.4. Evaluation for endophytic presence of B. bassiana in sorghum

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The endophytic establishment of B. bassiana in sorghum was tested in two ways – through stem culture and PCR. For stem culture, B. bassiana-treated

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sorghum stems were inoculated on Sabouraud dextrose yeast agar (SDAY)

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medium to facilitate growth of the fungus if present in the stem (Bing and Lewis

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1991). The PCR was set up with primers specific for fungal beta tubulin gene with DNA from sorghum plants treated with B. bassiana.

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To set up stem cultures, 10 plants were randomly selected from each replicate block in the treated and control twice: 30 and 60 d post-treatment. The plants were uprooted and the covering of leaf bases was peeled off to obtain the stem. Pieces of stem (1 cm) were cut at three regions (at different heights) of the stem (Table 1). For detection of B. bassiana at molecular level (PCR), two plants from

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each replicate block were selected at random for collecting stem material for DNA extraction.

2.4.1. Stem culture. SDAY medium was prepared with 0.01% of antibiotics chloramphenicol (0.05 mg ml−1) and streptomycin (0.05 mg ml−1) (to prevent

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bacterial contamination) and 0.1% crystal violet (to provide contrast for visualizing fungal growth (Chase et al. 1986)). The stem pieces were surfacesterilized first with 96% ethanol for 1 min, then with 6% sodium hypochlorite

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(NaOCl) solution for 5 min, followed by 96% ethanol for 30 s (Luginbuhl and Muller 1980). To ascertain the efficiency of the surface sterilization method,

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surface-sterilized stem pieces were imprinted onto SDAY plates. The absence of

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fungal or bacterial growth on these slants indicated the dependability of the sterilization technique (Ganley and Newcombe 2006). To set up culture, each

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stem piece was cut longitudinally with a sterile scalpel and plated on SDAY

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medium (4 pieces per culture slant) with the pith side touching the medium. The

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culture slants with inoculated stem pieces were incubated in an environmental chamber set at 25 ± 1 °C and 90% RH with no light. They were examined daily

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for 15 d. When fungal growth and conidia appeared from the stem piece, the conidia were mounted on a slide and examined under the microscope. When the conidial morphology resembled B. bassiana, the fungus on the stem pieces was inoculated in a new culture slant with SDAY medium with antibiotics to obtain a

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pure culture. The number of plants that showed endophytic presence of B. bassiana among the total tested was computed as a %.

2.4.2. PCR amplification using primers specific for fungal beta tubulin gene. DNA was extracted from B. bassiana-treated sorghum, untreated sorghum (negative

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control) and B. bassiana isolate ITCC 4688 used in treating sorghum (positive control) as described by Uma Devi et al. (2006). Primers for a conserved region of fungal beta tubulin gene – Bt2a-fwd: 5'-GTAACCA AATCGG

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TGCTGCTTTC-3' and Bt2b-rev: 5'ACCCTCAGTGTAGTGACCCTTGGC-3' (Glass and Donaldson 1995) were used. The PCR reaction medium was set up in a total

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volume of 25 µl. The reaction mixture consisting of 1µl of DNA (50 ng/µl), 2.5 µl

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of 10XPCR buffer with 15 mM MgCl2, 0.3 µl 5U Taq polymerase, 2.5 µl 2mM dNTPs (MetaBion, Martinsried, Germany), and 1µl (20 pM) each of the forward

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and reverse primers was made up to a final volume of 25 µl with sterile double-

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distilled water. PCR was carried out in a Mastercycler® ep Eppendorf thermo

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cycler (Eppendorf, Hamburg, Germany). The PCR programme was set as follows: an initial cycle of 94 °C for 3 min, followed by 30 cycles at 94 °C for 30 s,

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55 °C for 1 min and 72 °C for 30 s, followed by a final extension at 72 °C for 2 min. Following PCR amplification, an aliquot (5 µl) of PCR reaction mixture from each sample was electrophoresed on a 1% agarose gel with SYBR safe dye (Invitrogen, Germany). The gel was viewed on a transilluminator to identify

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samples with amplification products. The PCR-amplified products of one sample each from DNA of treated sorghum and B. bassiana were sequenced. For sequencing, the DNA of the amplified product was retrieved as follows. The PCR reaction mixture was run on a 1% agarose gel and the portion of the gel with the band (amplified product) was visualized on a dark reader (Clare Chemical

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Research, Colorodo, USA) and cut out. The DNA in the gel piece was purified using perfectprep® gel cleanup kit (Eppendorf, Hamburg, Germany) following the instructions of the manufacturer. The concentration of DNA in the purified

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PCR products from the gel was estimated spectrophotometrically. They were then sequenced in both directions. The sequencing reaction was set up using

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DNA of a volume equivalent to 140ng and 0.5 µl (10 pM) of the primer

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(forward/reverse) made up to a final volume of 6 µl with sterile double distilled water. Sequencing was done using BigDye® Terminator v 3.1 Cycle Sequencing

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Kit (PE Applied Biosystems Inc., Weiterstadt, Germany) on an automated

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Applied Biosystems 3730/xl/ 96-capillary DNA Analyzer (PE Applied

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Biosystems Inc., Weiterstadt, Germany). The sequences were compared to the sequences in GenBank database using BLASTn programme (Altschul et al. 1990)

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to identify homologous sequences.

2.5. Comparison of similarity of B. bassiana used for treatment of sorghum plants (ITCC 4688) and B. bassiana retrieved from sorghum stem cultures through AFLP fingerprinting

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B. bassiana has been reported to occur naturally as an endophyte in corn (Bing and Lewis 1991). To ascertain whether the B. bassiana retrieved from stem cultures of sorghum is a natural inhabitant or the artificially inoculated strain, DNA fingerprinting (AFLP) was done. The AFLP fingerprints of the B. bassiana isolate ITCC 4688 used for treating sorghum and three fungal cultures retrieved

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from B. bassiana-treated sorghum stem cultures, were generated as described in Uma Devi et al. (2006). Three primer sets were used – Pst-A/Mse-CAT, PstC/Mse-CAT and Pst-C/Mse-CTA. These primers were found to produce

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polymorphic bands and enable differentiation of isolates of B. bassiana (Uma Maheswara Rao 2004).

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2.6. Artificial infestation of sorghum plants with stem borer and assessment of protection from stem borer due to pre-treatment with B. bassiana

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In the second experiment , sorghum plants were treated with B. bassiana and

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later (after 15 d) infested with first instar larvae of C. partellus (30 larvae per

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plant) (Lewis et al. 2001). The sorghum plants not treated with B. bassiana but infested with stem borer larvae were taken to be positive controls for

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comparison. A block of sorghum plants treated with B. bassiana and a block of untreated sorghum plants not artificially infested with stem borer were taken as negative controls. The larvae were carefully placed in the whorl of the plant with a camel-hair brush. Twenty days after inoculation of larvae, 20 plants from each treatment block were randomly selected to check whether the artificially

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inoculated lab-bred larvae were successful in infesting the plants. The number of larvae in the leaf whorl and within the stem (examined by splitting open the stem) in each of the tested plants was noted. When the plants were 12-week-old and the panicles were at hard dough stage, 20 plants in each treatment block were uprooted. They were longitudinally split from the panicle to the base of the

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plant, and the extent of tunneling made by stem borer was measured as the number of tunnels and length of each tunnel within the stem. The number of plants with dead-hearts and those which did not bear panicles were counted in

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each block. The plant height and grain yield were measured as described above (in 2.4). The endophytic establishment of B. bassiana was checked through PCR

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randomly selecting two plants from each block.

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2.7. Statistical analysis

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The % values of plants that showed endophytic presence of B. bassiana among

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the total tested, plant height, yield, insects and tunnel length per plant were

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arcsine percent square root-transformed to normalize the data and the mean ± SE of each variable was back-transformed (Gomez and Gomez 1984). The statistical

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significance of the difference between B. bassiana-treated and control plants in the measured parameters was estimated through one way ANOVA. Statistical analysis was performed using Statistica software package (Stat soft Inc., 1995).

3. Results and Discussion

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No adverse effect on growth (as measured from plant height) and yield was observed in B. bassiana-treated plants when compared with the controls in the experiments carried out in the first season (Table 1). Thus, the relationship of B. bassiana with sorghum can be inferred as one of an endophyte and not a pathogen.

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“[Insert Table 1 about here]”

B. bassiana was detected in the stem cultures of treated plants but not in the control. B. bassiana was found in stem pieces collected from all heights of the

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plant tested (Table 2). B. bassiana is reported to move passively through xylem vessels, thereby spreading through the entire stem (Wagner and Lewis 2000).

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Endophytic establishment of the fungus was more frequent when plants were

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treated with aqueous conidial spray than when treated with conidiating fungal culture on a rice substrate (Table 2). In corn, no such difference in the

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two types of treatments was observed (Lewis et al. 2001). A few other fungi

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such as Fusarium spp. and Aspergillus spp. were also observed in stem

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cultures. In B. bassiana-treated sorghum stem cultures, Myrethecium roridum Tode was also found. The plants from which these stem cultures were

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initiated had brown spots on leaves. The brown spots could have been induced by M. roridum. M. roridum is known to cause brown leaf-spot disease in millets. The control plants (not treated with B. bassiana) which were in a separate plot did not show brown-spot symptoms. Unlike the infestation of the phytopathogenic brown spot fungus on B. bassiana-treated sorghum plants

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observed in our experiments, in tomato, seed treatment with B. bassiana was reported to confer resistance to phytopathogenic fungus Rhizoctonia solani Kühn that causes seedling wilt (Owenly et al. 2004). “[Insert Table 2 about here]” PCR using primers specific for fungal beta tubulin gene showed amplification

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of a 360 bp product both in B. bassiana and B. bassiana-treated sorghum plants but not in untreated sorghum (Figure 1). The PCR amplicons from the two sources – pure culture of isolate ITCC 4688 of B. bassiana and sorghum treated with ITCC

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4688 were found identical to each other in sequence. The sequences have been deposited in GenBank with accession numbers EU486431 and DQ784576.2

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respectively. The 360bp sequence showed 98% similarity with beta tubulin

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sequence of B. bassiana isolate BCC 14482 with an e value of 2e-161 and 96% similarity with beta tubulin gene of the B. bassiana isolate NRRL 22866 with an e

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value of 6e-161 and 98% similarity to five other isolates of B. bassiana with an e

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value of 4e-133. Some of the beta tubulin sequences of this region of B. bassiana

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isolates already available in the GenBank showed 100 and 99% similarity between them. B. bassiana is a species complex (Rehner and Buckley 2005).

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Isolates are ascribed to this species based on morphology. Being a predominantly asexual fungus, it is not possible to test for reproductive isolation between members identified as B.bassiana to differentiate between species. Perhaps, this could account for both: (i) the range of variation (6 %) in the conserved region of beta tubulin gene among the deposited accessions of this region from B. bassiana

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in GenBank, and (ii) the lack of 100% similarity of the B. bassiana isolate ITCC 4688 of our study with any other of the B. bassiana sequences in the GenBank. Using a similar molecular approach, B. bassiana could not be detected in B. bassiana-treated tomato seedlings though it was endophytically present (Ownley et al. 2004). In their experiment, universal (not fungus-specific) primers for rRNA

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genes (White et al. 1990) were used. In the B. bassiana-treated sorghum, an additional band of 746 bp was also observed (Figure 1). It has been deposited in GenBank and was assigned an

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accession number DQ871250. The 746 bp sequence, from B. bassiana-treated sorghum plants matched among other fungal sequences, a length of 18 bp region

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in Cytochrome P 450 gene of M. roridum. The sorghum plants in the B. bassiana-

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treated plot as mentioned earlier, showed brown spot symptoms caused by M. roridum.

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“[Insert Figure 1 about here]”

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AFLP fingerprints of all the three B. bassiana isolates randomly selected from

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among the several isolates obtained from stem culture of treated sorghum plants were found to be identical to the B. bassiana isolate ITCC 4688 used for treating

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sorghum plants with all the three primer combinations tested (Figure 2). The AFLP fingerprints of a few B. bassiana isolates (from Uma Maheswara Rao 2004) with the primer/adaptor combinations used in this study are also given in the figure demonstrating the polymorphism observed among them with these AFLP primers (Figure 2). Besides having identical AFLP fingerprints, the beta tubulin

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sequence amplified from the isolate ITCC 4688 (EU486431) and the sequence amplified from sorghum treated with ITCC 4688 (DQ784576.2) were also identical. Thus, we conclude that the B. bassiana isolated from artificially inoculated sorghum is the same isolate as the one used for artificial inoculation (ITCC4688) and not a naturally associated strain. Corn is reported to naturally

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harbour B. bassiana as an endophyte (Bing and Lewis 1991). “[Insert Figure 2 about here]”

The B. bassiana-treated plants artificially infested with larvae of stem borer (C.

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partellus), had significantly lesser number of larvae compared to the controls (not treated with B. bassiana) when examined 20 days after infestation (Table 3).

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Forty percent (40.06 ± 1.6) of the control plants that were artificially infested with

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C. partellus larvae dried up with dead-heart. In the surviving control plants, stem tunneling by larvae was 25% as compared to ~4% in B. bassiana-treated sorghum

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(Table 3). A similar observation has been reported in B. bassiana-treated corn.

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(Lewis et al. 1996). Among the surviving control plants, 36% (35.8 ± 1.9)

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produced a panicle, and even in these plants, plant height and grain yield was significantly less than the B. bassiana-treated plants (Table 3). The plants

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randomly checked through PCR in the second season for the presence of B. bassiana did show endophytic presence of B. bassiana. In the experiments performed in the second season, there was natural infestation of sorghum plants by aphids, army worm (Mythimna seperata Walker) and Helicoverpa armigera Hübner. The aphids were spotted on four-week-old

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seedlings at the time of treatment with B. bassiana. The aphid infestation disappeared in B. bassiana-treated plants within 5 d post-treatment while it persisted for 30 d in the control plants (not treated with B. bassiana). During later stages, the control plants (not treated with B. bassiana) also showed mild infestation of H. armigera and M. seperata while these insect pests were not

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noticed on B. bassiana-treated plants. The effect of the natural attack of these pests in control plants was reflected in their growth. The height and yield of these plants was significantly less than in the B. bassiana-treated sorghum plants (Table

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3).

“[Insert Table 3 about here]”

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B. bassiana treatment not only resulted in protection against the stem borer, but

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also decimated naturally infesting aphid population and prevented infestation by other insect pests such as army worm and H. armigera.

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Thus, treatment of sorghum at seedling stage with the entomopathogenic

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fungus B. bassiana can be used as an effective method for establishing the fungus

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endophytically as a means of protection from the plant intruding stem borer. Such treatment at early stage protects the plant also from other insect pests that

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infest at later stages of plant life.

Acknowledgements We thank the Andhra Pradesh Netherlands Biotechnology Unit, Institute of Public Enterprise, Osmania University Campus, Hyderabad, India for financial

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support (project number: BTU P-76). We thank DST (New Delhi; India) and DAAD (Germany) for financial support for collaboration under DST-DAAD PPP 03 programme INT/DAAD/P-89/2003. Narasimha Reddy P is thankful to APCOST, Hyderabad for a Young Scientist Fellowship. References

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Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol. 215: 403 − 410. Arnold AE, Lewis LC. 2005. Ecology and evolution of fungal endophytes and

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their roles against insects. In: FE Vega, Blackwell M, editors. Insect-fungal associations: Ecology and evolution. New York: Oxford University Press. p. 74 − 96.

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Bills GF, Polishook JD. 1991. Microfungi from Carpinus caroliniana. Can J Botany. 69: 1477 − 1482.

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Bing LA, Lewis LC. 1991. Suppression of Ostrinia nubilalis by endophytic

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Beauveria bassiana. Environ Entomol. 20: 1207 − 1211.

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Chase AR, Osborne LS, Ferguson VM. 1986. Selective isolation of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae from an

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artificial potting medium. The Florida Entomologist 69: 285 – 292.

Ganley RJ, Newcombe G. 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycol Res. 110: 318 – 327. Gethi M, Mutind AC, Diallo A. 2001. Stem borers in maize: A natural stress and progress towards host plant resistance. Proceedings of the 7th Eastern and

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Southern Africa Regional Maize Conference; 2001 Feb 11 -15; Nairobi. Kenya, 45 − 48 p. Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microb 61: 323 − 330.

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Gomez AK, Gomez AA. 1984. Statistical procedures for agricultural research. Singapore: John Wiley and son’s Inc. p. 643 − 645. Gomez Vidal S, Lopez Llorca LV, Jansson HB, Salinas J. 2006. Endophytic

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colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron 37: 624 − 632.

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Inglis GD, Goettel MS, Johnson DL. 1995. Influence of Ultraviolet Light

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Protectants of the Entomopathogenic Fungus, Beauveria bassiana. Biol Control 5: 581 − 590.

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Jenkins NE, Heviefo G, Langewald J, Lomer CJ. 1998. Development of mass

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production technology for aerial conidia for use as mycopesticides. Biocontrol

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News Infor. 19: 21 − 31.

Jones KD. 1994. Aspects of the biology and biological control of the European

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corn borer in North Carolina. [PhD thesis]. [Raleigh]: North Carolina State University. Leckie BM. 2002. Effects of Beauveria bassiana mycelia and metabolites incorporated in to synthetic diet and fed to larval Helicoverpa zea, and

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detection of endophytic Beauveria bassiana in tomato plants using PCR and ITS. [MS thesis]. [Knoxville]: The University of Tennessee. Lewis LC, Berry EC, Obrycki JJ, Bing LA. 1996. Aptness of insecticides (Bacillus thuringiensis and carbofuran) with endophytic Beauveria bassiana, in suppressing larval populations of the European corn borer. Agr Ecosyst

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Environ. 57:27–34.

Lewis LC, Bruck DJ, Gunnarson RD, Bidne KG. 2001. Assessment of plant pathogenicity of endophytic Beauveria bassiana in Bt. transgenic and non-

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transgenic corn. Crop Sci. 41: 1395 − 1400. Luginbuhl M, Muller E. 1980. Endophtische Pilze in den oberirdischen Organen

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von 4 gemeinsam an gleichen Standorten wachsenden Pflanzen (Buxus,

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Hedera, Ilex, Ruscus). Sydowia 33: 185 − 209.

Maniania NK. 1993. Evaluation of three formulations of Beauveria bassiana for

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control of the stem borer Chilo partellus. J Applied Entomol. 115: 266 − 272.

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Ownley BH, Pereira RM, Klingeman WE, Quigley NB, Leckie BM. Beauveria

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bassiana, a dual purpose biocontrol organism, with activity against insect pests and plant pathogens. In: Lartey RT, Caesar AJ, editors. Emerging concepts in

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plant health management, Research Signpost, Trivandrum, Kerala, India, 2004: 255 − 269. Posada F, Vega FE. 2005. Establishment of the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) as an endophyte in cocoa seedlings (Theobroma cacao). Mycologia 97: 1208 − 1213.

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Posada F, Vega FE. 2006. Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycoscience 47: 284 − 289. Posada F, Aime MC, Peterson SW, Rehner SA, Vega FE. 2007. Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota:

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Hypocreales). Mycol Res. 111: 748 − 757. Quesada-Moraga E, Landa BB, Mun˜oz-Ledesma J, Jime´nez-Dia´z RM, SantiagoA´lvarez Morga C. 2006. Endophytic colonisation of opium poppy, Papaver

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somniferum, by an entomopathogenic Beauveria bassiana strain. Mycopathologia 161: 323 − 329.

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Rehner SA, Buckley E. 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-

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sequences: evidence for cryptic diversification and links to

Cordyceps teleomorphs. Mycologia 97: 84-98.

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Tulsa OK: StatSoft, Inc., 2325 East 13th street, Tulsa, OK, 74104.

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Uma Devi K, Padmavathi J, Sharma HC, Seetharama N. 2001. Laboratory evaluation of the virulence of Beauveria bassiana isolates to the sorghum shoot

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borer Chilo partellus and their characterization by RAPD-PCR. World J Microb Biot. 17: 131 − 137. Uma Devi K, Reineke A, Nageswara Rao Reddy N, Uma Maheswara Rao C, Padmavathi J. 2006. Genetic diversity, reproductive biology, and speciation in

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the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin. Genome 49: 495 − 504. Uma Maheswara Rao C. 2004. Investigation of the genetic structure through DNA fingerprinting of an epizootic population of Nomuraea rileyi (Farlow) Samson and a worldwide population of Beauveria bassiana (Balsamo)

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Vuillemin and their virulence characterization. [PhD dissertation]. [Visakhapatnam, India]: Andhra University. Wagner BL, Lewis LC. 2000. Colonization of corn, Zea mays, by the

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entomopathogenic fungus Beauveria bassiana. Appl Environ Microbiol. 66: 3468 − 3473.

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White TJ, Bruns T, Lee S, Taylor JW. 1990. Amplification and direct sequencing of

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fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A guide to methods and

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applications. New York: Academic Press Inc. p. 315 − 322.

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Legends to figures

Figure 1. PCR products amplified using primers specific for a conserved region of the fungal beta tubulin gene (Glass and Donaldson 1995) from DNA of – 1: sorghum, 2: Beauveria bassiana, 3: sorghum treated with B. bassiana.

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Figure 2. AFLP fingerprints with three different primer combinations of Beauveria bassiana isolate ITCC 4688 used for treatment of sorghum (1) and B. bassiana

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isolates (2, 3, 4) retrieved from stem cultures of B. bassiana-treated sorghum. AFLP fingerprints of four B. bassiana isolates (5, 6, 7, & 8 NRRL 22864, NRRL

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22865, ARSEF 1149 & ITCC 4521 respectively) generated with the same enzyme,

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adaptor and primer combination (Uma Maheswara Rao, 2004) are displayed for comparison. M – DNA size marker.

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Table 1. Comparison of growth and yield of Beauveria bassiana treated and untreated (control) sorghum plants.

Treatment type Test

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Aqueous Conidial spray

30 days

53.6±0.7

Control

53.6±0.8

ANOVA‡

F1,2=0.012 P=0.92

Conidiating fungal culture on rice

Plant height(cm)* 60 days 90 days

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Yield (kg)†

30 days

Plant height(cm)* 60 days 90 days

127.4±1.6

203.6±7.3

4.20±0.05

60.8±1

135±1.0

203±2.7

4.17±0.5

130.9±1.4

208.3±4.3

4.12±0.07

60.5±1

137±1.2

207±2.1

4.30±0.18

F1,2=7.7 P=0.1

F1,2=16.55 P=0.56

F1,2=5.16 P=0.15

F1,2=0.22 P=0.67

F1,2=2.46 P=0.25

F1,2=9.54 P =0.09

F1,2=0.41 P =0.58

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Both the treated and the control plants showed three tillers. * Values †Mean ‡

Yield (kg)†

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represent mean ± SE of three replicate blocks with measurements of 20 plants from each.

yield of 20 plants; values represent mean± SE of three replicate blocks.

P value in all comparisons is insignificant.

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Table 2. Endophytic colonization of the entomopathogenic fungus Beauveria bassiana in sorghum stems. % Endophytic colonization * Treatment method Height of the stem from the base Aqueous conidial Spray Conidiating fungal culture on rice substrate ANOVA(between treatments) †

60th day post treatment

day post treatment

3cms

18cm

33cm

5cm

25cm

45cm

56.67± 0.96

71.69 ± 1.00

69.25 ± 2.04

65.86 ± 1.33

82.54 ± 1.09

76.73 ± 1.48

45.83 ± 0.47

61.67 ± 0.49

57.57 ± 0.83

48.33 ± 0.47

69.02 ± 1.36

64.17 ± 0.49

F 1,2=10.33 P = 0.03

F 1,2= 43.35 P = 0.02

F 1,2=163.18 P = 0.006

F 1,2=25.66 P = 0.007

F 1,2=26.63 P = 0.006

F 1,2=34.91 P = 0.027

were angular transformed (arc sine percentage) before analysis, back-

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* Values

30th

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transformed and rounded. Values represent mean ± SE of 20 plants from each of the three replicates. †

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P value is significant in all comparisons.

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Table 3. Comparison of effect of artificial infestation with larvae of stem borer in B.bassiana pretreated and control sorghum plants.

Fo

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Mean tunnel length (cm)/ plant* ---

60th day

90th day

18.38± 0.36

99.2±4.11

119.5±4.14

Mean no of insects/ plant* ---

18.31± 0.72

95.2±1.88

103.9±2.2

---

---

4.75±0.06

17.3± 0.67

63.5±1.18

82±1.9

---

---

3.55±0.15

ANOVA (df=2,4)Treated vs. control

F 2,4 =2.58, P= 0.19

F 2,4 =147‡

F 2,4 =855.1‡

C. partellus infested and aqueous conidial spray C. partellus infested and conidiating fungal culture on rice Control C. partellus infested

18.63± 0.43

89.7± 2.13

98.7±2.5

0.55±0.21

4.06±0.64

4.49±0.18

16.86± 0.62

91.9± 2.39

97.9±2.13

0.65±0.18

4.51±0.98

4.41±0.11

17.13± 0.66

39.5± 1.14

44.3±1.78

3.25±0.72

24.29±1.1

2.84±0.1

ANOVA (df =2,4) Treated vs. control

F 2,4 = 6.09, P = 0.06

F 2,4 =434‡

F 2,4 = 273.6‡

F 2,4 = 2514.1‡

F

F 2,4 = 44.14‡

Treatment type

Aqueous conidial spray

Conidiating fungal culture on rice Control

* Values †Mean

‡

Plant height (cm) * 30 days

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Yield (kg)† 4.72±0.13

F 2,4=39.3‡

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2,4 =

841.31‡

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represent mean ± SE of three replicate blocks with measurements of 20 plants from each.

yield of 20 plants; values represent mean± SE of three replicate blocks.

P value significant.

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