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Enhanced Resistance to Spodoptera litura in Endophyte InfectedCauliflower PlantsAuthor(s): Abhinay Thakur , Sanehdeep Kaur , Amarjeet Kaur , and Varinder SinghSource: Environmental Entomology, 42(2):240-246. 2013.Published By: Entomological Society of AmericaURL: http://www.bioone.org/doi/full/10.1603/EN12001

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INSECT-SYMBIONT INTERACTIONS

Enhanced Resistance to Spodoptera litura in Endophyte InfectedCauliflower Plants

ABHINAY THAKUR,1 SANEHDEEP KAUR,1,2 AMARJEET KAUR,3 AND VARINDER SINGH3

Environ. Entomol. 42(2): 240Ð246 (2013); DOI: http://dx.doi.org/10.1603/EN12001

ABSTRACT Endophytic fungi, which live within host plant tissues without causing any visiblesymptom of disease, are important mediators of plantÐherbivore interactions. These endophytesenhance resistance of host plant against insect herbivores mainly by productions of various alkaloidbased defensive compounds in the plant tissue or through alterations of plant nutritional quality. Twoendophytic fungi, i.e., Nigrospora sp. and Cladosporium sp., were isolated from Tinospora cordifolia(Thunb.) Miers, a traditional indian medicinal plant. Caulißower (Brassica oleracea L.) plants wereinoculated with these two endophytic fungi. The effect of endophyte infected and uninfectedcaulißower plants were measured on the survival and development of Spodoptera litura (Fab.), apolyphagous pest. Endophyte infected caulißower plants showed resistance to S. litura in the form ofsigniÞcant increase in larval and pupal mortality in both the fungi. Inhibitory effects of endophyticfungi also were observed on adult emergence, longevity, reproductive potential, as well as hatchabilityof eggs. Thus, it is concluded that antibiosis to S. litura could be imparted by artiÞcial inoculation ofendophytes and this could be used to develop alternative ecologically safe control strategies.

KEY WORDS fungal endophytes, Nigrospora sp., Cladosporium sp., Spodoptera litura, insectÐplantinteractions

Microorganisms living inside the host plant are im-portant mediators in plantÐherbivore interactions(Berenbaum 1988, Hammon and Faeth 1992). Amongthe microorganisms, the fungal endophytes livingwithin plant tissues without causing visible diseasesymptoms at a particular time (Stone et al. 2000) playan important role to confer resistance in plants againstherbivores by producing toxic metabolites, mainly al-kaloids (Clay 1990, Siegel and Bush 1996). A variety ofalkaloids like egrot alkaloids, loline alkaloids, lolitrems,and peramine have been reported from endophyteinfected grasses that are absent in noninfected con-speciÞcs (Bacon et al. 1986, Rowan et al. 1986). Thealkaloids produced by the fungus or by the plant inresponse to the fungal infection increase host resis-tance to herbivores (Powell and Petroski 1992, Mullerand Krauss 2005). Endophytes usually found to behighly toxic in agricultural systems (e.g., Neotypho-dium endophytes) may not be so in native speciesbecause of reduced diversity and quantity of alkaloidsproducedbyendophytes infectingnative speciescom-pared with the high levels produced in agriculturalgrasses (Faeth et al. 2002). A recent study indicatesthat the endophyte infection frequency of sleepygrassvaries from 50 to 100% in different geographic loca-tions and toxic alkaloid production in infected plants

varies from 0 to �150 �g g-1 tissue (Faeth et al. 2006).Endophytic fungi can also indirectly affect the herbi-vores by altering plant nutritional chemistry qualita-tively and quantitatively (Bernays 1993, Dugassa-Go-bena et al. 1996, Clay and Holah 1999). The deterrenceof insect herbivory depends on the fungal strain andgrowth stage of the plant (Tintjer and Rudgers 2006).These microorganisms have strong effects on the com-munity structure and diversity of associated organ-isms, e.g., bacteria, nematodes, and insects (Omaciniet al. 2001).

The previous research in temperate regions showedthat endophyte infected grasses have been reported toinhibit the growth and development of the feedinginsects (Afkhami and Rudgers 2009, Crawford et al.2010). Colonization of an endophytic fungus Neoty-phodium sp. in meadow fescue and ryegrass deterredfeeding and adversely affected the development ofSpodoptera frugiperda (J.E. Smith) (Ball et al. 2006).Similarly, Brem and Leuchtmann (2001) documentedsigniÞcantly lower weights, delayed pupation and amuch higher mortality in S. frugiperda larvae fed onEpichloe infected leaves of Brachypodium sylvaticum(Huds.) Beauv. as compared with those fed on unin-fected leaves. The larval growth of Popillia japonicaNewman beetle larvae also inhibited in Neotypodiumsp. infectedTaraxacumofficinale(Weber)(Richmondet al. 2004). In addition, Sabzalian et al. (2004) re-ported a signiÞcant suppression of population growthof mealybugPhenococcus solaniFerris, and aphidSiphamaydis Passerini, on fungal endophyte infected tall

1 Department of Zoology, Guru Nanak Dev University, Amritsar(Punjab), India, 143005.

2 Corresponding author, e-mail: sanehsaini@gmail.com.3 Department of Microbiology, Guru Nanak Dev University, Am-

ritsar (Punjab), India, 143005.

0046-225X/13/0240Ð0246$04.00/0 � 2013 Entomological Society of America

and meadow fescues. The plants artiÞcially inoculatedwith endophytes also showed resistance to insect her-bivores. Tomato (Solanum lycopersicum L.) plants in-oculated with unspecialized soil borne endophyteAc-romonium strictum Gams inßuenced the preferenceand performance of greenhouse whiteßies (Vidal1996). Similarly, a signiÞcant increase in larval mor-tality of Plutella xylostella (L.) occurred because offeeding on cabbage plants inoculated with Acremo-nium alternatum. Recently, Hernawati et al. (2011)also demonstrated the role of leaf endophytic fungi ofchili (Capsicum annuum L.) in protection againstAphis gossypii Glover.

However, most of the existing information on en-dophytes comes from temperate hosts, especially fromgrasses.Onlya fewstudies areavailableonendophytesfrom tropical plants, although they have a great di-versity of these microorganisms. In light of this, thepresent studies were conducted to measure the anti-insect potential of endophytic fungi from tropicalplants.

Endophytic fungi were isolated from Tinospora cor-difolia (Thunb.) Miers. It is indigenous to the tropicalareas of India, Myanmar, and Sri Lanka and is a richsource of alkaloids, terpenoids, and phenolic com-pounds (Singh et al. 2003, Phan et al. 2010). Takingadvantage of the potential of endophyte infectedgrasses as biocontrol agents the present studies wereconducted to measure the effect of endophyte in-fected caulißower plants on Spodoptera litura (Fab.),which is one of the major pest of many economicallyimportant crops like cruciferous vegetables, cucurbits,groundnut, and cotton (Rao et al. 1994, Qin et al.2004). It has also developed physiological resistance tomost of the commercial available insecticides.

Materials and Methods

Insect Rearing. Initial stocks for the culture of S.litura were obtained from the caulißower Þeldsaround Amritsar (Punjab), India. The subsequentgenerations were reared under controlled tempera-ture and humidity conditions of 25�C and 70%, re-spectively. The rearing was carried out in battery jars(15 � 10 cm) on castor leaves with daily change ofdiet. The pupae were transferred to pupation jarscontaining 2Ð3 cm layer of moist sterilized sand cov-ered with Þlter paper. The adults on emergence wereshifted to oviposition jars similar to pupation jars ex-cept for a cotton swab soaked with honey solution(one part honey: four parts water) as food, hangingfrom the muslin cloth covering the jar. The ovipositionjars were lined with Þlter paper to facilitate egg laying.For experimental studies, the newly hatched larvaewere transferred to artiÞcial diet as recommended byKoul et al. (1997) with slight modiÞcations.Isolation of Endophytic Fungi. The stems and

leaves of healthy plants of T. cordifolia growing in thecampus of Guru Nanak Dev University, Amritsar(Punjab), India, were used for isolation of endophyticfungi. The respective materials were washed thor-oughly using distilled water, followed by washing with

70% ethanol for 2 min and 5% sodium hypochloritesolution for 5 min to accomplish surface sterilization.The treated samples were rinsed in sterile distilledwater before plating. The samples were cut into 5Ð6pieces 2Ð6-mm size and were plated on water agarplates (distilled water, 1.5% (wv�1) agarÐagar) sup-plemented with ampicillin (HiMedia, Mumbai, India)(200 �g ml�1) as antibacterial agent. The plates wereincubated at 30�C for 3Ð4 d. The hyphal tips thatemerged from the plant parts were picked, puriÞed,and maintained on PDA (Potato Dextrose Agar)plates for further studies. Isolated fungi were identi-Þed as Nigrospora sp. (Fig. 1A) and Cladosporium sp.(Fig. 1B) on the basis of morphological charactersGilman (2001).Inoculation of Cauliflower Plants. Caulißower be-

ing a preferred host of S. litura was selected to studythe effect of endophytic fungi on this insect. Cauli-ßower seedlings were transplanted in pots Þlled withmixture of soil and vermicompost in the ratio of 4:1.The pots were kept in glasshouse chamber at 20Ð22�C.For inoculating caulißower plants, spore suspensionswere prepared from 3-wk-old fungal cultures. In eachslant, 10 ml of distilled water and one drop of 0.01%Tween 80 (HiMedia, Mumbai, India) were added toget a homogenous suspension and spore count wasobtained with the help of hemocytometer. Theplants were inoculated separately with 150 ml ofspore suspension consisting of 2.50 � 106 spores/mlof Nigrospora sp. and 2.75 � 106spores/ml of Cla-dosporium sp.; control plants were treated with 150ml of water containing 0.01% Tween 80.

In each treatment, Þfty plants were taken and singleplant replicate of inoculated and control plants wereplaced randomly in glass house chamber. After theinoculation, stemand leaf sampleswerecollectedafter3 wk from six randomly selected plants of both treat-ments, and to assess endophyte infection, fungi werereisolated as explained previously in isolation method.If at least 20% of sample segments showed endophytegrowth, the inoculated plants were considered as suc-cessfully infected and ready for use in the experiments(Jallow et al. 2008). All experiments were conducted3 wk after inoculation.Bioassay Studies.Three weeks after inoculation, the

older leaves lying on the lower parts of endophyteinoculated and endophyte free plants were used forthe experimental purpose. The second-instar larvae ofS. litura were fed on leaves of inoculated and endo-phyte free plants. The larval rearing was carried inbattery jars (15 � 10 cm) at 27�C temperature and 60%RH. Each experiment was replicated eight times with10 larvae per replication. To ensure hygenic condi-tions, leaves were changed regularly and battery jarswere washed daily. Observations were made daily onvarious biological parameters viz. larval and pupalmortality, larval and pupal period, adult emergence,adult deformity, fecundity, and percent hatching.

All the values were represented as their mean � SE.To compare difference in means, one way analysis ofvariance with TukeyÕs test (P� 0.05) was performed.SPSS software for windows version 16.0 SPSS Inc,

April 2013 THAKUR ET AL.: RESISTANCE TO S. litura IN ENDOPHYTES 241

Chicago and Microsoft OfÞce Excel 2003 MicrosoftCorp., USA were used to perform the statistical anal-ysis.

Results

The fungi isolated from T. cordifoliawere identiÞedasNigrospora sp. andCladosporium sp. The caulißowerplants artiÞcially infected with these fungi did notshow any disease symptoms. The plants vigor of en-dophytes infected plants also did not differ from en-dophytes free plants. Both the endophytic fungi, i.e.,Nigrospora sp. and Cladosporium sp. had a signiÞcantinhibitory effect on performance of S. litura. Larvaefeeding on leaves from infected plants were compar-atively sluggish to control larvae. The larvae suffered31.25 and 30% mortality because of Cladosporium sp.and Nigrospora sp., respectively, as compared with7.50% in control (F 2,21 � 6.20, P � 0.05). Larvaesurvived on endophyte infected caulißower plants fur-ther suffered signiÞcantly higher pupal mortality inboth the fungi (F 2,21 � 23.91, P � 0.05) (Fig. 2A).

The larval mortality started after 10 and 13 d offeeding on Nigrospora sp. and Cladosporium sp. in-fected plants, respectively, with maximum number oflarval deaths on the 17Ð18 d of exposure. However, incase of the control, larval mortality did not occur untilafter 17 d of feeding (Fig. 2B). Most of the larvae diedat the time of molting to the last instar. The larvaereared on leaves of plants infected with Cladosporiumsp. took signiÞcantly longer time (F 2,21 � 22.22, P �0.05) to pupate as compared with those on uninfectedplant but the pupal period did not differ signiÞcantly.Nigrospora sp. infected plants did not signiÞcantlyinßuence the larval development but the pupae de-veloped from these larvae took signiÞcantly more time

(F 2,21 � 9.13, P � 0.05) to eclose to adults Fig. 2C).SigniÞcantly fewer number of adults emerged fromlarvae reared on Nigrospora sp. and Cladosporium sp.infected plants than on uninfected plants (F 2,21 �50.29,P� 0.05) (Fig. 2D). Some of the adults exhibitedmorphological deformities like crumpled and unequalwings as compared to normal adults (Fig. 3A). Suchmalformed adults lived for a very short time period. Asis evident from (Fig. 2D) the number of malformedadults was signiÞcantly higher when larvae werereared on Cladosporium sp. infected plants (F 2,21 �7.95, P� 0.05). Inhibitory effects of endophytic fungialso were observed on adult longevity, reproductivepotential, as well as hatchability of eggs. The life spanof females that emerged from larvae fed on Cladospo-rium sp. infected plants reduced signiÞcantly as com-pared with control (F 2,6 � 10.61, P� 0.05) (Fig. 4A);however, male longevity remained unaffected. Thefemales emerged from larvae reared on leaves fromNigrospora sp. infected plants were found to be sig-niÞcantly less fecund (F 2,6 � 7.07, P � 0.05) as com-pared with control adults. However,Cladosporium sp.treated plants did not signiÞcantly inßuence the fe-cundity (Fig. 4B). The toxic effect of endophytic fungiwere observed further on egg hatchability, which de-creased signiÞcantly (F 2,6 � 23.09, P � 0.05) by 8.20and 6.27%, respectively, because ofNigrospora sp. andCladosporium sp. over control (Fig. 4C).

Discussion

The results of present studies demonstrated signif-icant inhibitory effects of endophytic fungi on survivaland development of S. litura. Caulißower plants in-fected with Cladosporium sp. and Nigrospora sp. iso-lated from T. cordifolia induced signiÞcantly higher

Fig. 1. (A) Morphology of Nigrospora sp. and (B) Cladosporium sp. (Online Þgure in color.)

242 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 2

larval mortality in S. litura than those feeding on un-treated plants. The development of S. litura larvaesigniÞcantly prolonged because of feeding on leaves ofcaulißower plants infected with Cladosporium sp.,whereas the pupal development was signiÞcantly pro-longed on plants infected with Nigrospora sp.

Adverse effects of endophytes have been reportedearlier on different species of Spodoptera and otherinsect pests, but most of the previous work has cen-tered uponAcremonium spp. from cool season grasses.Recent studiesdemonstrated thatethyl acetateextractof endophytic Nigrospora sp. signiÞcantly inhibitedthe larval survival, adult emergence, longevity, andreproductive potential of S. litura (Thakur et al. 2012).Similar to our Þnding, Hernawati et al. (2011) alsoreported anti-insect potential of Nigrospora sp. iso-lated from chili against A. gossypii. A signiÞcant in-crease in mortality of the greenhouse whiteßy larvaefeeding on tomato plants inoculated with A. strictumhas been documented earlier by Vidal (1996). Simi-larly, the larvae of P. xylostella also suffered signiÞ-cantly higher mortality because of feeding on cabbageplants inoculated with A. alternatum (Raps and Vidal1998). Endophyte infected leaves of caulißower plantsfurther reduced adult longevity, fecundity, and via-bility of eggs of S. litura. Likewise,H. armigera larvae,when reared on endophyte inoculated tomato plants,suffered signiÞcant reduction in growth rate, pro-longed development time, suppressed molting, and

produced smaller pupae that were more likely to die.The adults emerged from pupae were less fecund ascompared with control (Jallow et al. 2003). Contraryto this, Raps and Vidal (1998) did not detect signiÞcantdifference in length of development period, adult sur-vival time, and number of eggs when P. xylostellalarvae were reared on leaves of cabbage plant inocu-lated with A. alternatum as compared with controlplant. Similarly, Faeth and Hammon (1997) found nocorrelation of endophyte infected leaves with in-creased leaf minor mortality on oak. Leuchtmann(1992) suggested that when the endophytes were ar-tiÞcially inoculated with in nonhost plants, some ofthem were effective, whereas others were not. How-ever, in the present studies the endophytes got asso-ciated with the caulißower plants as is evident frombioassay studies and both the endophytic fungi werereisolated from the infected plants.

A signiÞcant number of pupae and adults emergingfrom larvae fed onCladosporium sp. andNigrospora sp.infected caulißower plants showed growth deformi-ties comparable to those which can be elicited byjuvenile hormone analogous in insect diets (Nes et al.1997). Several studies have demonstrated that growthand molting of insect larvae can be negatively affectedby changing the phytosterol composition of the diets(Richter et al. 1987). Cholesterol uptake might also beinhibited by metabolites produced by microorganismsand result in reduced availability of dietary sterol

Fig. 2. Effect of endophyte infected and uninfected plants on survival and development of S. litura. (A) Larval mortalityand pupal mortality; (B) mortality distribution of S. litura larvae reared on endophyte infected and uninfected caulißowerplants; (C) larval period, pupal period; (D) adult emergence and deformities. Means and SE are given. Means within a columnfollowed by the same letter are not signiÞcantly different (P � 0.05) based on TukeyÕs test.

April 2013 THAKUR ET AL.: RESISTANCE TO S. litura IN ENDOPHYTES 243

(Bernays 1993) which are essential for the synthesis ofmolting hormone. Most of the larval deaths of S. liturafeeding on endophyte infected plants occurred at thetime of molting which may be because of change insterol concentration. Dugassa-Gobena et al. (1996)demonstrated that sterol proÞle of tomato plants in-oculated with A. strictum altered both qualitativelyand quantitatively. This variability in certain sterols inendophytic plants may not only change the nutritional

quality of the plants for herbivorous insects (Bernays1993) but may also account for reduced growth andfood conversion efÞciency.

The mechanism resulting in retarded growth anddevelopment of S. litura when larvae fed on leaves ofplants infected with Cladosporium sp. and Nigrosporasp. is not known with certainty, but it may be becauseof secondary compounds produced by the endophyteor by the host in response to the endophyte. Julia

Fig. 3. (A,B,C) deformed adults and (D) normal adults. (Online Þgure in color.)

Fig. 4. (A) Male and female longevity of S. litura larvae fed on leaves of caulißower plants infected with endophytic fungiNigrospora sp. and Cladosporium sp. and untreated control leaves, (B) fecundity, and (C) hatching. Means within a columnfollowed by the same letter are not signiÞcantly different (P � 0.05) based on TukeyÕs test.

244 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 2

(2010) reported the presence of alkaloids, quinones,and phenolics compounds in ethyl acetate extract ofendophyte Nigrospora sp. isolated from the mangroveplant Bruguiera sexangula (Lour.) Poir. Cladosporiumsphaerospermum isolated from the mangrove plantAe-giceras corniculatum (L.) Blanco also yielded com-pounds of same category. These compounds havebeen reported to have repellent and antifeedant ac-tivity against S. litura (Morimoto and Komai 2006).The caulißower plants associated with Cladosporiumsp. and Nigrospora sp. showed antibiosis resistance toS. litura in the form of increased larval and pupalmortality, prolonged development time, and morpho-logical deformities. As the endophyte infected plantsneither showed any disease symptoms nor any varia-tion in their growth, so the adverse effects on insectperformance may be because of change in physiologyof endophyte infected plants.

Acknowledgments

We thank The Head, Department of Zoology, and Head,Department of Microbiology, Guru Nanak Dev University,Amritsar (Punjab), India for providing all necessary facilitiesduring the course of study. Financial assistance from Uni-versity Grants Commission, Government of India, NewDelhi, is duly acknowledged.

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Received 3 January 2012; accepted 9 January 2013.

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