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Detrimental effects of endophyticfungus Nigrospora sp. on survival anddevelopment of Spodoptera lituraAbhinay Thakur a , Sanehdeep Kaur a , Amarjeet Kaur b & VarinderSingh ba Department of Zoology , Guru Nanak Dev University , Amritsar ,Indiab Department of Microbiology , Guru Nanak Dev University ,Amritsar , IndiaAccepted author version posted online: 08 Dec 2011.Publishedonline: 28 Feb 2012.

To cite this article: Abhinay Thakur , Sanehdeep Kaur , Amarjeet Kaur & Varinder Singh (2012)Detrimental effects of endophytic fungus Nigrospora sp. on survival and development of Spodopteralitura , Biocontrol Science and Technology, 22:2, 151-161, DOI: 10.1080/09583157.2011.646952

To link to this article: http://dx.doi.org/10.1080/09583157.2011.646952

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RESEARCH ARTICLE

Detrimental effects of endophytic fungus Nigrospora sp. on survival anddevelopment of Spodoptera litura

Abhinay Thakura, Sanehdeep Kaura*, Amarjeet Kaurb and Varinder Singhb

aDepartment of Zoology, Guru Nanak Dev University, Amritsar, India; bDepartment ofMicrobiology, Guru Nanak Dev University, Amritsar, India

(Received 3 August 2011; final version received 1 December 2011)

Endophytes have been known to confer resistance to host plants against insectherbivores mediated by fungal alkaloids. In this study we have isolated anendophytic fungus Nigrospora sp. (Ascomycota: Sordariomycetes) from a nativeplant Tinospora cordifolia. To assess anti-insect potential, we tested to what extentthe survival and development of Spodoptera litura (Lepidoptera: Noctuidae), apolyphagous pest, was affected when fed on artificial diet amended with differentconcentrations of ethyl acetate extract of Nigrospora sp. In feeding assay, theinsect suffered significantly higher mortality and showed prolonged developmentperiod on amended diet as compared to unamended diet. A significant reductionin relative growth and consumption rate, efficiency of conversion of ingested anddigested food and approximate digestibility indicated deterrent as well as toxiceffects of the fungal extract. Significant adverse effects were also observed onadult emergence, longevity and reproductive potential of S. litura. This pre-liminary information on insecticidal properties of Nigrospora sp. may further beused for imparting resistance in plants against insects.

Keywords: fungal endophytes; Nigrospora sp.; Spodoptera litura; fungal extract;insect plant interactions

1. Introduction

Among the various microorganisms, endophytic fungi are known to impart

resistance to host plants against various insect pests (Clay 1989). Endophytes are

commonly defined as fungi or bacteria that live asymptomatically within healthy

plant tissues for at least a part of their life cycle (Wilson 1995; Stone, Bacon, and

White 2000; Evans, Holmes, and Thomas 2003). Webber (1981) was probably the

first to report the role of endophyte, Phomopsis oblongata in protecting elm trees

against the beetle Physocnemum brevilineum. Similar observations on the control of

insect pests such as sod webworm, argentine stem weevil and fall armyworm by

endophytic fungi have been reported by other workers (Funk et al. 1983; Gaynor and

Hunt 1983; Clay, Hardy, and Hammond 1985; Hardy, Clay, and Hammond 1985).

The mechanism underlying the anti-insect properties of endophytes is attributed to

the production of various alkaloid-based defensive compounds in the plant tissue

(Clay and Holah 1999; Faeth 2002). Endophyte-infected grasses contain a variety of

alkaloids such as ergot alkaloids, loline alkaloids, lolitrems and peramine, which are

*Corresponding author Email: sanehsaini@gmail.com

Biocontrol Science and Technology,

Vol. 22, No. 2, February 2012, 151�161

ISSN 0958-3157 print/ISSN 1360-0478 online

# 2012 Taylor & Francis

http://dx.doi.org/10.1080/09583157.2011.646952

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absent in non-infected conspecifics (Bacon, Lyons, Porter, and Robbins 1986; Rowan

and Gaynor 1986). The alkaloids have been detected in liquid cultures of fungi

(Ahmad, Govindarajan, Funk, and Johnson-Cicalese 1985; Plowman, Leuchtmann,

Blaney, and Clay 1990). Taking advantage of the potential of endophyte-infectedgrasses, Clay (1989) had raised the possibility of using them as biocontrol agents.

Most of the reports available in literature pertaining to evaluation of anti-insect

properties of endophytes are from grasses in temperate regions (Saikkonen, Faeth,

Helander, and Sullivan 1998; Wilson 2000); relatively little is known of the nature of

the interactions between woody plants and their endophytes particularly from

tropical regions (Gilbert, Chang, and Rojas 2002; Van Bael et al. 2005). Thus there is

a need to explore the indigenous endophytes of commercially important plants for

their ability to induce resistance against insect pests. In light of this, endophytes havebeen isolated from Tinospora cordifolia, a traditional Indian medicinal plant which is

known by the common name guduchi. It is a herbaceous vine of the family

Menispermaceae indigenous to the tropical areas of India, Myanmar and Sri Lanka.

T. cordifolia is a rich source of alkaloids, terpenoids and phenolic compounds (Singh

et al. 2003; Phan et al. 2010).

The present studies were conducted to evaluate the potential of endophytic fungi

from T. cordifolia to control Spodoptera litura which is the most destructive pest of

different crops such as cruciferous vegetables, cucurbits, groundnut, cotton, maize,potato, soybean, tobacco and some pulses (Rao, Rajasekhar, Venkataiah, and Rao

1994; Qin, Ye, Huang, Ding, and Luo 2004). Although earlier reports indicated a

decrease in fall armyworm larval weight and survival, due to feeding on tall fescue

(Festuca arundinacea) infected with Acremonium spp. (Hardy, Clay, and Hammond

1986; Bultman and Conard 1998), no information is available on effect of endophytes

from T. cordifolia on S. litura. Thus the present study was undertaken to evaluate the

role of endophytes of T. cordifolia as biocontrol agent against S. litura.

2. Materials and methods

2.1. Insect rearing

Initial stocks for the culture of S. litura were obtained from the cauliflower fields

around Amritsar, Punjab, India, and subsequent generations were reared under

controlled temperature and humidity conditions of 258C and 70%, respectively. The

rearing was carried out in battery jars (15 cm�10 cm) on castor leaves with dailychange of diet. The pupae were transferred to pupation jars containing 2�3 cm layer

of moist sterilised sand covered with filter paper. The adults on emergence were

shifted to oviposition jars similar to pupation jars except for a cotton swab soaked

with honey solution (1 part honey:4 parts water) as food, hanging from the muslin

cloth covering the jar. The oviposition jars were lined with filter paper to facilitate

egg laying. The newly hatched larvae were transferred to fresh castor leaves.

2.2. Isolation of endophytic fungi

The stems and leaves of healthy T. cordifolia plants growing in their natural habitat in

Guru Nanak Dev University campus, Amritsar, India, were used for isolation of

endophytic fungi. The respective materials were thoroughly washed and disinfested

152 A. Thakur et al.

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using distilled water, followed by 70% ethanol for 2 minutes and 5% sodium

hypochlorite solution for 5 minutes. The treated samples were rinsed in sterile

distilled water prior to plating. The samples were cut into 5�6 pieces (2�6 mm size)

and were placed on water agar plates (distilled water, 1.5% (w v�1) agar�agar)

supplemented with ampicillin (200 mg ml�1). The plates were incubated at 308C for

3�4 days and observed for growth around the samples. The endophytic fungi which

emerged from the plated plant parts were isolated and maintained on Potato

Dextrose Agar (PDA) plates. Twelve endophytic cultures were isolated and further

evaluated for insecticidal potential.

2.3. Production of ethyl acetate extract

Plugs were cut from the periphery of 4�5 day old colonies on PDA with a 8 mm steel

borer. Three plugs were used to inoculate 50 ml of malt extract broth (malt

extract �20 g l�1, dextrose �20 g l�1, peptone �1 g l�1, pH �5.5) in 250 ml

Erlenmeyer flasks. The flasks were incubated on a rotary shaker (250 rpm) at 308Cfor 10 days after which the medium was extracted twice with 50 ml of ethyl acetate at

110 rpm at 408C. The extracts were concentrated using Rotavapour and dissolved in

1 ml of High Pressure Liquid Chromatography (HPLC) grade water and stored at

48C for further use.

2.4. Bioassay studies

An artificial diet was prepared according to Koul et al. (1997) which included wheat

bran, kidney bean flour, yeast, methyl-para-benzoic acid, ascorbic acid, sorbic acid,

etc. The ethyl acetate extracts of isolated endophytic fungi at a concentration of

1 ml g�1 were added to the diet. The second instar larvae were fed on these amended

as well as control diets and observations were made on their survival. Among the

tested endophytic cultures, only TC25 induced mortality in S. litura larvae. This

culture was identified as Nigrospora sp. (Ascomycota: Sordariomycetes) on

morphological basis (Larone 2002) (Figure 1), whereas there were no attempts to

identify the other endophytic isolates showing no insecticidal activity. Only

Nigrospora sp. was selected for further studies.

2.5. Effect of Nigrospora sp. on S. litura

To evaluate the effect of Nigrospora sp. on growth and development of S. litura, the

artificial diet was supplemented with five concentrations of ethyl acetate extract of

the fungus (C1� 0.75 ml g�1, C2 �1 ml g�1, C3� 1.25 ml g�1, C4� 1.5 ml g�1,

C5� 2 ml g�1). Then second instar larvae were reared on amended and unamended

diets at 258C temperature and 70% relative humidity (RH). Each experiment was

replicated six times with 10 larvae/replication. To avoid cannibalism, the larvae were

kept individually in plastic containers (4 cm�6 cm) and diet was changed regularly.

Daily observations were made on larval and pupal mortality, larval and pupal

period, adult emergence, adult longevity, fecundity and egg viability, and sublethal

effects such as larval, pupal and adult deformities.

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2.6. Nutritional analysis

The effect of ethyl acetate extract of Nigrospora sp. on food utilisation of second

instar larvae of S. litura was carried out following the procedure of Koul, Singh,

Singh, and Singh (2005) and compared with unamended diet for 6 and 12 days. The

larvae starved for 3�4 hours were weighed individually and released in plastic

containers (4 cm�6 cm) containing known amount of control and treated diets.

Each set of experiment was carried out using 10 larvae. Observations every 72 hours

were made on larval weight, residual diet and faecal matter and overall change in

each variable was compared with the last recorded value. At the end of each

experiment, i.e. after 6 and 12 days, dry weight of the larvae was determined by

incubating at 608C for 72 hours. Similarly dry weight of diet and faecal matter were

recorded to determine the loss of water under experimental conditions. The data

obtained were used to determine the nutritional indices on dry weight basis as

proposed by Waldbauer (1968).

Relative growth (RGR) and consumption rates (RCR) were calculated as G/I

(G�change in larval dry weight/day and I�starting larval dry weight; change in

larval dry weight is the difference in final larval dry weight/day and initial larval dry

weight) and C/I (C�change in diet dry weight/day and I�starting larval dry weight;

change in diet dry weight is the difference in final diet dry weight/day and initial

larval dry weight), respectively. Both are calculated as mg mg�1 d�1. Index of food

conversion efficiency (ECI) was calculated as 100�G/C; where G�dry weight gain

of insect and C�dry weight of food consumed. Approximate digestibility (AD) and

efficiency of conversion of digested food (ECD) were calculated as C � F/C�100

(where C�change in diet dry weight /day and F�dry weight of frass/day) and

G/C�F�100 (where G�change in larval dry weight/day, C�change in diet dry

weight/day and F�dry weight of frass/day), respectively. Efficiency of conversion of

ingested food (ECI), AD and ECD were calculated as per cent.

Figure 1. Morphology of Nigrospora sp.

154 A. Thakur et al.

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2.7. Statistical analysis

All the experiments were replicated six times and values were represented as their

mean9SE. To compare difference in means one way analysis of variance (ANOVA)

with Tukey’s test at P50.05 was performed. SPSS software for windows version 16.0

(SPSS Inc., Chicago) and Microsoft office Excel 2003 (Microsoft Corp., USA) were

used to perform the statistical analysis.

3. Results

A significant effect of ethyl acetate extract of Nigrospora sp. was observed on survival

and development of S. litura. Diet amended with various concentrations of the ethyl

acetate extract induced higher larval mortality as compared to control (Figure 2a)

but significant differences were observed only at 1.25 ml g�1 of extract where larvaesuffered 37% mortality as compared to 3% on unamended diet (P�0.005). Most of

the larvae died at the time of molting (Figure 3a). More than 20% pupal mortality

Figure 2. (a�d) Larval mortality, pupal mortality, larval period and pupal period

of Spodoptera litura larvae fed with diet containing different concentrations (C1�0.75 ml g�1, C2� 1 ml g�1, C3� 1.25 ml g�1, C4� 1.5 ml g�1 and C5� 2 ml g�1) of

ethyl acetate extract of Nigrospora sp. and unamended diet. Means and SE are given. Means

within a column followed by the same letter are not significantly different (P50.05) based on

Tukey’s test.

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was recorded when larvae were fed on diet having 1.25, 1.5 and 2 ml g�1 of crude

extract but the significant differences were recorded only at highest concentration

(P�0.011) (Figure 2b). The amended diet significantly prolonged the larval period

by 5�7 days as compared to unamended diet (P�0.00) (Figure 2c) whereas pupal

development remained unaffected (Figure 2d).

Addition of crude extract to artificial diet resulted in decrease of adult

emergence, longevity and fecundity, but viability of eggs remained unaffected

(Table 1). The average adult emergence decreased by 14.7�24.2% at concentrations

ranging between 1.25 and 2 ml g�1 but significant effect was observed only at the

highest concentration. Nigrospora sp. also influenced the life span of adults (Table 1).

Except for lowest concentration, longevity of males emerged from larvae fed on

amended diet reduced significantly by 2�4.3 days as compared to control. Similarly

the life span of females decreased from 8.5 days in control to 4.5�6 days on

supplemented diet. The short life span subsequently affected reproductive potential

of adults emerged from larvae fed on amended diet. The diet supplemented with

2 and 1.5 ml g�1 of ethyl acetate extract of Nigrospora sp. significantly reduced the

fecundity of adults as compared to control (P�0.001).The sublethal effects of fungal extract were evident from the emergence of

deformed adults from larvae fed on amended diet. These adults had crumpled and

unequal wings (Figure 3b and c) as compared to normal adults (Figure 3d). The

larval rearing on diet supplemented with 2 ml g�1 led to morphological deformities in

28% of adults which were significantly higher than control (Table 1).

Figure 3. (a) Larval mortality at the time of moulting, (b and c) morphologically deformed

adults and (d) normal adults.

156 A. Thakur et al.

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3.1. Food utilisation

Ethyl acetate extract of Nigrospora sp. reduced the food utilisation of the larvae

(Table 2). Amended diet resulted in 33�56% reduction in RGR and 30�46% in RCR

over unamended diet after 6 days of feeding. The larvae fed on diet supplemented

with 1.25 ml g�1 showed more than 50% reduction in their ECI and ECD over

control larvae. All the concentrations significantly reduced the consumption rate as

compared to control (P�0.00), but significant differences in growth rate were

recorded only at 1.25 ml g�1 (P�0.022). The same concentration further

significantly reduced the ECI and ECD of larvae as compared to control (ECI,

P�0.001; ECD, P�0.00). The AD of diet amended with 1 and 1.25 ml g�1

concentrations of fungal extract also decreased significantly as compared to

unamended diet (P�0.002) (Table 2). Similar results were obtained after 12 days

of feeding on diet supplemented with organic extract of Nigrospora sp. Prolonged

exposure of larvae on amended diet significantly reduced the growth rate whereas no

significant effect was observed on RCR as compared to control except for

1.25 ml g�1. Significant reduction in ECI and ECD was recorded at concentrations

1.25�1.5 ml g�1 and 1.25�2 ml g�1, respectively (ECI, P�0.011; ECD, P�0.003).

When compared with control, significant decrease in AD was observed in diet

amended with 1�2 ml g�1 of organic extract (P�0.00) (Table 3). These results

indicate that concentrations of crude extract of Nigrospora sp. ranging between 1.25

Table 2. Effect of ethyl acetate extract of Nigrospora sp. on growth, feeding and food

utilisation of S. litura larvae after 6 days of feeding. Means followed by different superscript

letters within a column are significantly different. Tukey’s test, P 50.05.

Treatment

RGR

(mg mg�1 d�1)

RCR

(mg mg�1 d�1)

ECI

(%)

ECD

(%)

AD

(%)

Control 1.290.22a 10.190.42a 12.390.69a 14.790.19a 84.691.62a

C1 0.890.04ab 7.090.80b 10.990.87a 14.591.14a 82.793.21a

C2 0.790.08ab 5.490.65b 10.691.21a 10.691.48ab 66.694.09b

C3 0.590.05b 5.790.33b 5.590.35b 6.390.42b 66.595.77b

C4 0.790.06ab 5.990.33b 9.291.76ab 12.292.10a 71.891.58ab

C5 0.790.15ab 5.990.75b 10.490.44a 13.290.51a 76.492.47ab

Table 1. Influence of different concentrations of ethyl acetate extract of Nigrospora sp. on

S. litura adults and their reproductive potential. Means followed by different superscript

letters within a column are significantly different. Tukey’s test, P 50.05.

Treatment

Adult

emergence

(%)

Adult

Deformity

(%)

Adult longevity (days)

Fecundity

(eggsfemale�1)

Hatching

(%)Male Female

Control 97.692.38a 2.192.08a 8.090.57a 8.590.57a 952924.96a 9791.13a

C1 93.094.46ab 4.292.63ab 6.090.00ab 6.090.28b 774943.69ab 9691.27a

C2 92.293.63ab 7.293.28ab 5.090.57b 5.590.76b 763964.86ac 8496.03a

C3 83.094.25ab 14.195.16ab 4.390.33b 5.090.50b 727967.55ac 9291.05a

C4 76.597.98ab 19.697.97ab 4.390.66b 4.790.33b 604959.63bc 9090.42a

C5 73.496.02b 28.598.89b 3.790.88b 4.590.28b 532921.90c 8693.03a

Biocontrol Science and Technology 157

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and 2 ml g�1 adversely affected food utilisation efficiency of S. litura. The reduced

values of RGR, RCR, ECI, ECD and AD indicated deterrent as well as toxic effectof the fungal extract.

4. Discussion

Our results demonstrate adverse effects of Nigrospora sp. on S. litura. Artificial diet

amended with this fungal extract led to reduced survival and growth of S. litura. The

addition of 1.25 ml g�1 of the extract to diet induced significantly higher larval

mortality as compared to control whereas significantly higher pupal mortality was

recorded at 2 ml g�1. The higher concentrations induced delayed toxic effects.

Although no studies are available on this endophytic toxin, reports on the effect of

other toxins demonstrated that larval survival of Spodoptera frugiperda was

significantly lower at 100 mg L�1 of egrocryptine and 10 mg L�1 of elymoclavinethan control (Clay and Cheplick 1989). Methanol extract of endophyte-infected tall

fescue seeds and stems induced significant mortality in aphids within 66 hours of

feeding (Johnson et al. 1985). Dowd, Cole, and Vesonder (1988) also documented

100 and 38.7% mortality, respectively, in S. frugiperda and Heliothis zea larvae fed on

artificial diets amended with fungal metabolites, roseotoxin B at 25 ppm while

dihydroxyaflavinine at the same concentration caused 20% mortality in both the

insect larvae. Shiba and Sugawara (2009) also documented reduced survival of

rice leaf bug, Trigonotylus caelestialium on artificial diet amended withN-formylloline (50 mg g�1) extracted from Neotyphodium uncinatum-infected

meadow fescue.

The development of S. litura was adversely affected by the crude extract of

Nigrospora sp. In contrast to the controls, larval development was signifi-

cantly delayed but pupal period remained unaffected. Similarly, the findings of

Clay et al. (1985) documented that fall armyworm, S. frugiperda larvae fed on leaves

of Cyperus virens infected with the endophyte Balansia cyperi took 35.5 days to

pupate as compared to 24.7 days for larvae fed on uninfected leaves. The negativeeffects on growth and development apparently resulted in reduced adult emergence,

longevity and fecundity. Although the diet amended with ethyl acetate extract of

Nigrospora sp. adversely affected the survival and development of S. litura, the

concentrations ranging from 1.25 to 2 ml g�1 were found to be the most effective.

Table 3. Effect of ethyl acetate extract of Nigrospora sp. on growth, feeding and food

utilisation of S. litura larvae after 12 days of feeding. Means followed by different superscript

letters within a column are significantly different. Tukey’s test, P 50.05.

Treatment

RGR

(mg mg�1 d�1)

RCR

(mg mg�1 d�1)

ECI

(%)

ECD

(%)

AD

(%)

Control 0.690.11a 1.790.06a 12.290.38a 21.493.94a 69.592.98a

C1 0.390.04b 1.690.05a 10.390.64ab 15.890.96ab 57.691.97ab

C2 0.290.02b 1.590.18a 9.690.94ab 12.891.79ab 55.393.77bc

C3 0.190.01b 1.390.07b 8.791.25b 11.791.19b 50.691.85bc

C4 0.290.02b 1.490.11a 7.490.41b 10.790.75b 46.894.06bc

C5 0.290.01b 1.690.10a 9.791.11ab 9.791.56b 44.492.93c

158 A. Thakur et al.

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The insignificant differences between these concentrations indicate that the effect of

organic fungal extract lies within this range.

The inhibitory effects on survival as well as on growth and development of

S. litura indicate toxicity of ethyl acetate extract of Nigrospora sp. which is further

evidenced by reduced food utilisation by larvae. There was decrease in relative

consumption and growth rate of larvae fed on amended diet. The prolonged

exposure of larvae on amended diet could not significantly influence the consump-

tion rate but as compared to control RGR of larvae was more reduced after 12 days

than 6 days of feeding. The ECI and ECD to insect biomass also decreased

significantly after 12 days at concentrations ranging between 1.25 and 2 ml g�1

except for ECI at 2 ml g�1. It is likely that decrease in consumption is due to anti-

feedant nature of the extract which further resulted in decreased growth rate. A drop

in ECI indicates that more food is being metabolised for energy required for

detoxification and less is being converted to insect biomass. As the proportion of

digested food metabolised for energy increased, ECD decreased. The efficiency with

which ingested and digested food is converted to insect biomass varies with its

nutritional value, digestibility and level of intake (Slansky 1993). This indicates that

food quality diminished by endophytic fungi and food converted to biomass with a

lower ECI and ECD. Larvae that suffered reduced ECD in relation to increased

metabolic cost may be able to increase their RCR in an attempt to compensate

(Slansky 1993), as demonstrated by Raps and Vidal (1998) that diamondback moth,

Plutella xylostella larvae feeding on endophyte-infected cabbage plants responded to

reduced ECI by increasing their relative consumption rate. However, in our studies,

RCR as well as ECI and ECD of S. litura decreased on amended diet which indicates

both deterrent as well as toxic effects of endophytes. This reduction in ECI and ECD

values resulted in early as well as delayed effects on S. litura population such as

deformities in adults, reduced adult longevity and fecundity. Delayed toxic effects

observed at 2 ml g�1 may be due to difference in activity of detoxification enzymes. It

is possible that these enzymes may initially be induced at this concentration

but prolonged exposure may have suppressed their activity and resulted in more

toxicity.

A study conducted by Riedell, Kieckhefer, Petroski, and Powell (1991) revealed

that methanolic extracts of F. arundinacea infected with Acremonium coenophialum

contain lolines of fungal origin which induced weight loss and behavioural

alterations in fall armyworm, S. frugiperda and European corn borer, Ostrinia

nubilalis when added to diet. In addition to increased mortality, Raps and Vidal

(1998) also observed reduced RGR and ECI of surviving larvae of P. xylostella

feeding on leaves of endophyte-infected cabbage plants. When crude extract of

Trichilia americana was incorporated into artificial diet of S. litura, it caused

significant reduction in RGR, RCR, ECI and ECD (Wheelar and Isman 2001).

Similar results were also seen with hirtin and Trichilia connaroidae extract when

tested against Peridroma saucia and S. litura (Xie et al. 1994).Although we have not conducted quantitative and qualitative analysis of

secondary compounds produced by Nigrospora sp., our nutritional analysis revealed

the anti-feedant and anti-biotic effects of crude extract. This study provides evidence

that Nigrospora sp. isolated from T. cordifolia possesses anti-insect properties and

may play an important role in protecting plants against insect pests.

Biocontrol Science and Technology 159

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Acknowledgements

Authors are thankful to The Head, Department of Zoology and Head, Department ofMicrobiology, Guru Nanak Dev University, Amritsar (Punjab), India, for providing allnecessary facilities during the course of study. Financial assistance from University GrantsCommission (UGC), Government of India, New Delhi, is duly acknowledged.

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