Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These...

6
55 INTRODUCTION MATERIALSAND METHODS Survey: Isolation of the fungus: Morphological studies and identification: Ophiocordyceps Ophiocordyceps Fungi Ascomycota Pezizomycotina Sordariomycetes Hypocreomycetidae Hypocreales Ophiocordycipitaceae et al. Cordyceps viz Cordyceps Ophiocordyceps Metacordyceps Elaphocordyceps C. sinensis Ophiocordyceps O. sinensis. Cordyceps cord ceps et al Ophiocordyceps Ophiocordyceps Ophiocordyceps Leocopholis coneophora Ophiocordyceps Ophiocordyceps Ophiocordyceps . is one of the most valued and best known fungal genus in traditional Chinese Medicine. Even though the stromatal heads of resembles mushrooms, it is an ascomycetous entomopathogenic fungus (Kingdom , Phylum , Subphylum , Class , Subclass , Order , and Family ). Based on molecular phylogenetic study, Sung (2007) separated the mega genus into four genera, . , , and . As a result, members like and related organisms were transferred to the genus , hence renamed as The name comes from two Latin words: and , meaning "club" and "head", respectively (Holliday and Cleaver, 2004). It parasitizes the body of the insect host and utilizes the internal tissues as a substrate for its growth, followed by complete replacement of internal tissues by mycelium, which finally get converted into a sclerotium. Later from the sclerotial bodies, stroma of the fungus emerges above the ground, thus it is commonly called as “Caterpillar fungus”. It is collected along with the sclerotium for medicinal and other uses (Li ., 2011). Due to strict parasitism and specific geographic environment in which it grows, the output of natural cannot meet market demand and thus its price is unusually high. In the Indian markets this fungus is valued as much as Rs. 15 lakhs/kg in recent years. Most of the bioactive compounds of sp. have been exploited for use in traditional and modern ethno-medicine, for treatment of various diseases like cancer, diarrhoea, headache, muscle pain and so on. From the surveys conducted in the coastal sandy areas of Kasargod district, it was observed that entomopathogenic species of was emerging during southwest monsoon (June-August) from the infected coconut white root grub ( Burm.), which is an endemic pest in the coastal sandy tracts of Kasragod District (Kumar and Aparna, 2014). So far there are no systematic studies conducted on the mycological aspects of sp. from Kerala and hence a detailed investigation was carried out focusing on the isolation, identification and characterization of sp. Surveys were carried out for the occurrence of fruit bodies of in Padannakkad, Valiyaparamba and Pilicode area of Kasargod district of Kerala, during June to August months of 2017 and 2018. Observations of the fruit bodies were made The fruit bodies were collected in brown paper covers and brought to the laboratory for further observations. These fruit bodies were preserved fresh as well as in dry forms. Isolation of fungus was carried out from different parts of the fungal structures like sclerotia, stipe and stroma. The isolation was performed by following the standard tissue culture technique. The samples were washed in running tap water. From all the three parts (sclerotia, stipe and stroma), tissues were taken for isolation. These tissues were cut into small pieces using a sterile blade, and subsequently cut small pieces were disinfected with mercuric chloride (0.1%) for one minute. After three washings using sterilized distilled water, the samples were placed on solidified potato dextrose agar (PDA) medium aseptically. After incubation at room temperature (26 ± 2 C), the fungal growth from second to sixth days of incubation was subsequently sub-cultured to solidified PDA in sterile Petri dishes. Purification of isolate was achieved by periodic sub- culturing and maintenance of the isolate was done in PDA slants under refrigerated condition at 4 C for further studies. Morphological studies (macroscopic and microscopic) of the fungus were o o KAVAKA53: 55-60(2019) P. K. Laya *, C. K.Yamini Varma , K.Anita Cherian , M. M.Anees and C. R. Rashmi Observations on entomopathogenic fungus on Coconut root grub Ophiocordyceps neovolkiana Leucopholis 1 2 3 4 5 1,3,4 2 5 Department of Plant Pathology, College of Horticulture, Vellanikkara 680656, Kerala, India Department of Plant Pathology, AICRP on Spices, Pepper Research Station, Panniyur 670142, Kerala, India Department of Vegetable Science, AICVIP, College of Horticulture, Vellanikkara 680656, Kerala, India *Corresponding authorEmail: [email protected] Submitted onAugust 20, 2019;Accepted on November 19, 2019) ABSTRACT Ophiocordyceps Ophiocordyceps O. sinensis Cordyceps Ophiocordyceps Leucopholis coneophora Ophiocordyceps neovolkiana are entomopathogenic fungi on arthropods which parasitize, kill and mummify hosts and produce fruit bodies out of them. Due to the presence of many bioactive compounds like adenosine, cordycepin, ergosterol, these fungi have been used as food as well as medicine against many diseases. Even though cosmopolitan in distribution, many like are mostly confined to high Himalayan Mountains in India, China, Tibet, Nepal, Bhutan at an altitude of 3000 to 5000 msl. Owing to high price, are known as "Himalayan gold". From surveys conducted in coastal sandy areas of Kasargod District (Kerala), it was observed that sp. attacks coconut root grubs ( Burm.), which is an endemic pest in the sandy tracts. The fungus was isolated in potato dextrose agar medium. Detailed morphological studies have been carried out. The molecular characterization showed homology with (Kobayasi) strain KC1 with 98% identity. The ITS sequence has been deposited at NCBI with accession # MH668282 and culture has been deposited in the National Fungal Culture Collection of India with herbarium # NFCCI 4331. KEYWORDS: Cordyceps, isolation, morphology, coconut root grub, coastal sandy region, southwest India, Kasargod coneophora Adoi:10.36460/Kavaka/53/2019/55-60

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Page 1: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

55

INTRODUCTION

MATERIALSAND METHODSSurvey

Isolation of the fungus

Morphological studies and identification

Ophiocordyceps

Ophiocordyceps

Fungi AscomycotaPezizomycotina SordariomycetesHypocreomycetidae HypocrealesOphiocordycipitaceae

et al Cordycepsviz Cordyceps Ophiocordyceps

Metacordyceps ElaphocordycepsC sinensis

Ophiocordyceps O sinensisCordyceps cord ceps

et al

Ophiocordyceps

Ophiocordyceps

Ophiocordyceps

Leocopholis coneophora

Ophiocordyceps

Ophiocordyceps

Ophiocordyceps

is one of the most valued and best knownfungal genus in traditional Chinese Medicine Even thoughthe stromatal heads of resemblesmushrooms it is an ascomycetous entomopathogenic fungus(Kingdom Phylum Subphylum

Class Subclass Order and Family

) Based on molecular phylogeneticstudy Sung (2007) separated the mega genusinto four genera

and As a result memberslike and related organisms were transferred to thegenus hence renamed as Thename comes from two Latin words and meaning club and head respectively (Holliday andCleaver 2004) It parasitizes the body of the insect host andutilizes the internal tissues as a substrate for its growthfollowed by complete replacement of internal tissues bymycelium which finally get converted into a sclerotiumLater from the sclerotial bodies stroma of the fungus emergesabove the ground thus it is commonly called as ldquoCaterpillarfungusrdquo It is collected along with the sclerotium formedicinal and other uses (Li 2011) Due to strictparasitism and specific geographic environment in which itgrows the output of natural cannot meetmarket demand and thus its price is unusually high In theIndian markets this fungus is valued as much as Rs 15lakhskg in recent years Most of the bioactive compounds of

sp have been exploited for use in traditionaland modern ethno-medicine for treatment of various diseaseslike cancer diarrhoea headache muscle pain and so on

From the surveys conducted in the coastal sandy areas ofKasargod district it was observed that entomopathogenicspecies of was emerging during southwestmonsoon (June-August) from the infected coconut white root

grub ( Burm) which is an endemicpest in the coastal sandy tracts of Kasragod District (Kumarand Aparna 2014) So far there are no systematic studiesconducted on the mycological aspects of spfrom Kerala and hence a detailed investigation was carriedout focusing on the isolation identification andcharacterization of sp

Surveys were carried out for the occurrence of fruitbodies of in Padannakkad Valiyaparambaand Pilicode area of Kasargod district of Kerala during Juneto August months of 2017 and 2018 Observations of the fruitbodies were made The fruit bodies were collected in brownpaper covers and brought to the laboratory for furtherobservations These fruit bodies were preserved fresh as wellas in dry forms

Isolation of fungus was carried outfrom different parts of the fungal structures like sclerotiastipe and stroma The isolation was performed by followingthe standard tissue culture technique The samples werewashed in running tap water From all the three parts(sclerotia stipe and stroma) tissues were taken for isolationThese tissues were cut into small pieces using a sterile bladeand subsequently cut small pieces were disinfected withmercuric chloride (01) for one minute After threewashings using sterilized distilled water the samples wereplaced on solidified potato dextrose agar (PDA) mediumaseptically After incubation at room temperature (26 plusmn 2 C)the fungal growth from second to sixth days of incubation wassubsequently sub-cultured to solidified PDA in sterile Petridishes Purification of isolate was achieved by periodic sub-culturing and maintenance of the isolate was done in PDAslants under refrigerated condition at 4 C for further studies

Morphologicalstudies (macroscopic and microscopic) of the fungus were

o

o

KAVAKA53 55-60 (2019)

P K Laya C KYamini Varma KAnita Cherian M MAnees and C R Rashmi

Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkianaLeucopholis

1 2 3 4 5

134

2

5

Department of Plant Pathology College of Horticulture Vellanikkara 680656 Kerala IndiaDepartment of Plant Pathology AICRP on Spices Pepper Research Station Panniyur 670142 Kerala IndiaDepartment of Vegetable Science AICVIP College of Horticulture Vellanikkara 680656 Kerala IndiaCorresponding author Email mail2layapkgmailcomSubmitted onAugust 20 2019Accepted on November 19 2019)

ABSTRACTOphiocordyceps

Ophiocordyceps O sinensisCordyceps

OphiocordycepsLeucopholis coneophora

Ophiocordyceps neovolkiana

are entomopathogenic fungi on arthropods which parasitize kill and mummify hosts and produce fruit bodies out of them Dueto the presence of many bioactive compounds like adenosine cordycepin ergosterol these fungi have been used as food as well as medicineagainst many diseases Even though cosmopolitan in distribution many like are mostly confined to highHimalayan Mountains in India China Tibet Nepal Bhutan at an altitude of 3000 to 5000 msl Owing to high price are known asHimalayan gold From surveys conducted in coastal sandy areas of Kasargod District (Kerala) it was observed that spattacks coconut root grubs ( Burm) which is an endemic pest in the sandy tracts The fungus was isolated in potatodextrose agar medium Detailed morphological studies have been carried out The molecular characterization showed homology with

(Kobayasi) strain KC1 with 98 identity The ITS sequence has been deposited at NCBI with accession MH668282 and culture has been deposited in the National Fungal Culture Collection of India with herbarium NFCCI 4331

KEYWORDS Cordyceps isolation morphology coconut root grub coastal sandy region southwest India Kasargod

coneophora

Adoi1036460Kavaka53201955-60

carried out Various characters like shape size and colour ofsclerotia stipe and stroma and branching pattern of the stromaassociated with the insect-fungal complex were studied andobservations were recorded

Microscopic slides were prepared using lactophenol cottonblue stain and observed under compound microscope at 5x10x 40x and 100x magnifications to study the anamorphicstage including hyphae synnemata conidiophores andconidia and teleomorphic stage including perithecia asciand ascospores of the fungus Measurements andphotomicrographs were taken using Zeiss Axiolab imageanalyzer

Molecular characterization of the isolate was done to identifythe isolate at species level by DNA barcoding using universalprimers of ITS sequences Sequence analysis and nucleotidehomology of the fungus was analysed through the BLASTprogramme of NCBI (http ncbinlmnhmgovblast)Sequence analysis of the culture showed homology with

having 98 per cent identity Thesequence of the fungus was deposited at the GenBank of NCBIwithaccessionnumberMH668282and theculturewasdepositedin National Fungal Culture Collection of India at MACSAgharkarInstitutePunewithculturenumberNFCCI4331

Purposive sampling surveys were conducted in threelocations of coastal sandy tracts of Kasargod district duringJune to September months of 2017 and 2018 The fruitingbodies of sp emerged from the parasitized grubwere collected from three locations Instructional Farm ofCollege of Agriculture Padannakkad Valiyaparamba area ofNileshwar and Regional Agricultural Research StationPilicode where the coconut root grub is an endemicpolyphagous pest

The fruit bodies were found to be emerged from the soil aftersummer showers usually during mid May and after the onsetof monsoon showers during early June The population offruit bodies in one square meter area of soil were assessed inall the three locations and represented in the Amongthe three areas surveyed maximum number of fruiting bodies(average of 1386 m ) were found at Instructional Farm ofCollege ofAgriculture Padannakkad Kasargod

The fungus was isolated on PDAmedium and namedasCD1( )andwas brought intopurecultureThemyceliaappeared initially white later turning to creamish white tosalmon colour ( ) and the underside of the plate beinglight brown ( ) Subsequently numerous thread likepinkish synnemata appeared in the culture representing theanamorphic stage sp The anamorph producedsimilar type ofconidiaas produced in thefield

The fruit body of the fungus wasfound to be emerging from the cephalic region of the body ofthird instar grub of The club-shaped upper portion of fruit body called as stroma which isthe fertile portion in which the perithecia were present themiddle stalk-like part stipe and the mummified dead grubknown as sclerotium were observed ( ) The stipe andstroma portion of the fruiting bodies were found to be abovethe soil and infected grub was located at a depth of around 5 to10 cm below the soil ( )Average length and girth of thefruiting bodies obtained from all the three locations weremeasured ( ) in which the maximum length of 119 cmand 16 cm for stipe and stroma respectively were obtainedfrom Instructional farm of College of AgriculturePadannakkad Maximum girth of 39 cm and 17 cm for stipeand stroma was also observed from the same soil ( )The longitudinal section of the fruiting body showedcomplete mycelial colonization of the insect body by thefungus where the internal organs of the grub were replacedwith fungal mycelium ( )

Comprehensive study on the process of fruit body emergencewas carried out in all the three locations From the buriedinfected grubs yellowish-orange coloured stalks of about03-06 cm thickness emerged during the last week of May orfirst week of June and the tip of which later became bulgedand turned to bright orange red in colour Stromatal headswere observed to emerge from the cephalic region of theinfected third instar grubs of After 40 days bythe second week of July the stroma region showed numeroushumps which were initially orange red in colour turning tobrown representing formation of perithecia The colourturned to dark-brown with the complete maturation ofperithecia and the release of ascospores occurred in a periodof two weeks During August after an interval of two weeksthe stroma region became dark-brown to black and numerouswhite colored hook like sporulating structures calledsynnemata were developed representing the conidial stage of

Ophiocordyceps neovolkiana

Cordycepsviz

Hirsutella

Leucopholis coneophora

L coneophora

RESULTSSurvey

Table 1

IsolationFig 1a

Fig 1bFig 1c

Morphological studies

Fig 1d

Fig 1e

Table 2

Fig 1f

Fig 1g2

Number of fruiting bodies m2Mean2017 2018

Padannakkad 1525 1250 1386Valiaparamba 575 450 513

Pilicode 275 225 250

Table 1 Extent of fruit bodies in three locations (mean of fourreplications)

Fig1aFig1bampcFig1dFig1eFig1fFig1g

Typical colonies of sp on isolation plateCultureplate of spAssociationof spwith

sp in naturalhabitatVariationin sizeof fruitbodyL Sof sp

OphiocordycepsOphiocordyceps

Ophiocordyceps Leucopholis coneophoraOphiocordyceps

Ophiocordyceps

56 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Fig 3aFig 3bFig 3c

Fig 3dFig 3eFig 3fFig 3g

Hypha of spSynnemataDifferent stages of development of synnemata on thefruiting body in natural habitatSingle spherical conidia on conidiophoresSpherical conidiaSpindle conidia in chain on conidiophoresSpindle conidia

Hirsutella

Description Dimension (microm)

Hypha Hyaline septate and branched 11- 152

Synnemata Cylidrical with numerous conidiophores 2136 -2351 times 991-1100

Conidiophore Elongated single or branched 564-878

Spherical conidia Born singly on conidiophore 19-57

Spindle conidia Born in chains on conidiophore 25-63 times 053-241

Table 3 Microscopic characters of anamorphic stage

the fungus ( sp) ( ) Hence a gradual changeof colour and texture of stroma was seen during differentstages of development ( ) Branching of stroma wasnoticed as double and very rarely three to five branches from asingle stroma were observed ( ) After one month bythe second week of September the whole fruiting body gotdecomposed by the action of soil dwelling saprophytesVarious other macroscopic characters like shape of the fruitbody colour of sclerotium stipe and stroma collected fromthe three locations surveyed were also studied and theobservations are shown in

Microscopic observations ofsp such as characters of anamorphic stage

including hyphae synnemata conidiophores and conidia andteleomorphic stage including perithecia asci and ascosporeswere taken

Hyphae were hyaline septate and branched with a width of 11to 152 microm ( ) The length and width of synnematafrom which the conidiophores were arising was found to be2136 to 2351 microm and 991 to 1100 microm respectively ( )Synnemata was found to have cylindrical shape withnumerous conidiophores all over the surface In naturalhabitat the synnemata started to form initially as whitecottony cushion later turning to light pinkish white hook likestructures on the stroma having a length of 02-13 cm long( )

The conidia were formed either singly or in chains on longconidiophores measuring 564 to 878 microm Theconidiophores can be single or branched The conidia weredimorphic and hyaline among which one type of the conidia

was spherical with a diameter of 19 to 57 microm ( ) andthe other was spindle shaped with a length of 25 to 63 micromand width of 053 to 241 microm ( ) The sphericalconidia were arranged singly ( ) and spindle shapedconidia were arranged in chains on the conidiophores (

)

Microscopic examination of thin cross sections of stromashowed numerous perithecia towards the peripheral regions( ) They were peripheral to slightly immersedglobular to oval in shape narrow towards the tip ( )Openings of perithecia the ostioles through which releaseof ascospores to the outside occurs were found at the tip( ) The length and width of perithecia was found to be826 to 3961 microm and 936 to 1713 microm respectively Thethickness of the perithecial wall ranged from 166 to 217microm ( ) On the examination of perithecianumerous elongated asci were observed inside theperithecium Crozier formation stage during the ascusdevelopment was observed in immature perithecium (

) On maturation asci were observed as a bundle insidethe perithecium ( ) The ascus was elongated withlength ranging from 1154 to 1701 microm and width of 52 to10 microm ( ) The tip of the ascus had a prominent cap oroperculum the lid-like structure through which theascospores are released out of ascus ( ) The ascuspossesses eight filiform ascospores ( ) which werefour-partite with 1053 to 1357 microm length and 25 microm to 27microm width ( ) The ascospores usually split into four

Hirsutella

Ophiocordyceps

Fig 2a

Fig 2b

Fig 2c

Table 2

Microscopic studies

Fig 3a

Fig 3b

Fig 3c

Fig 3e

Fig 3gFig 3d

Fig3f Table 3

Fig 4aFig 4b

Fig 4c

Figs 4def g

Fig5a

Fig 5b

Fig 5c

Fig 5dFig 5e

Fig 5f

Characters of fruiting bodyShape Size (cm) Colour

Length Girth Sclerotia Stipe SromaStipe Stroma Stipe Stroma

Padannakkad Spindle 119 39 16 17 Black Dark-brown

Bright orange turningto dark-brown

Valiaparamba Club 85 28 13 14 Dark-brown

Dark-brown

Light-brown turningto dark-brown

Pilicode Club 104 32 12 14 Dark-brown

Brown Reddish-brownturning to dark-brown

Table 2 Macroscopic characters of sp in three locations(mean of 15 replicates)

Cordyceps

Fig 2aFig 2bFig 2c

Different stages of fruiting body development in the fieldChanges in stroma during developmentBranching pattern of stroma

57P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

Fig 5aFig 5bFig 5cFig 5dFig 5eFig 5fFig 5g

Crozier formation during ascus developmentMass of mature asciAsciAscus with operculumEight ascospores in an ascusAscosporesPart spores of ascospore

Description Diamension (microm)

Perithecia Globular -oval with ostiole 826-3961 times 936-17127

Perithecial Wall Thick 166-2165

Ascus Elongated 1154-17011 times 52-10

Acsospore Filiform and four partite 1053-1357 times 251-273

Ascospore partspore

Terminal spore pointed at the tip and middlespores are cylindrical

2558-4459 times 252-274

Table 4 Microscopic characters of teleomorphic stage

part spores in which the middle two spores werecylindrical and the terminal spores were pointed at the tiphaving 2558 to 4459 microm length and 252-274 microm width( )

a well-known and valued traditionalChinese medicinal mushroom also called Dong Chong XiaCao (winter worm summer grass) is a parasitic complex offungus and caterpillar of family

Due to the presence of many bioactive compoundslike adenosine cordycepin ergosterol havebeen used as food as well as in herbal medicine to treatrespiratory pulmonary renal liver and cardiovasculardiseases hyposexuality and hyperlipidemia (Arora 2015)Even though the distribution of this fungus is cosmopolitanmany of its members like are mostly confined tohigh Himalayan Mountains in China Tibet Nepal Bhutan andIndia at an altitude of 3000 to 5000 msl (Sharma 2004) Butfrom the surveys conducted in costal sandy tracts of KasargodDistrict of Kerala it was found that spparasitizes the third instar grubs of The present observations are in accordance with the report of(Kumar and Aparna 2014) Sreedhar and Karun (2017)reported the presence of associatedwith sap-sucking pentatomid stinkbug inWestern Ghats Dattaraj (2018) documented reports byvarious scientists about the occurrence of and alliedspecies in the Western Ghats and southwest coast of India andalso reported six species of recovered in the scrubjunglesof southwest Karnataka

So far there are no systematic studies conducted on themycological aspects of sp from Kerala andtherefore this study is a first attempt for the isolation andmorphological characterization of this fungus The period ofoccurrence of sp as per the samplingsurveys conducted in three selected locations of Kasargoddistrict during 2017 and 2018 has similarity with reports ofother scientists in different countries It was reported that thebest time of collection ofspecimens is from July to September in Japan (Kobayasi andShimizu 1980) and Korea (Nam 2006) The presence ofthe fungus in tropical conditions at low altitudes of Keralamay be attributed to the high humidity and low temperaturesituations prevailing in the state during south-west monsoonperiods the time at which the fungus was noticed in the field(Kumar andAparna 2014)

In the fruiting bodies of sp the bulged tipportion was observed as stroma and stalk like structure asstipe and sclerotium was the mummified grub which was

Fig 5g Table 4DISCUSSIONOphiocordyceps sinensis

Hepialis armoricanusHepialidae

Ophiocordyceps

O sinensis

OphiocordycepsLeucopholis coneophora

Ophiocordyceps nutansHalyomorpha halys

et alCordyceps

Cordyceps

Ophiocordyceps

Ophiocordyceps

Cordyceps sphecocephala

et al

Ophiocordyceps

Fig4aFig 4bFig 4cFig 4dFig 4eFig 4fFig 4g

StromaPeritheciaOstioleC S of stromaPeripheral arrangement of peritheciaGlobular to oval peritheciaMicroscopic characters of stroma

58 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

completely filled with mycelia of the fungus The tips ofperithecia were found as numerous humps which are initiallyorange red in colour turning to dark-brown These findingswere having similarity with observations of other researchersThe morphological characters of was described byShrestha (2010) as it consists of two parts a fungalendosclerotium (caterpillar) and stroma which was initiallyyellow in colour and later turning to dark brown or blackwhich was longer than the caterpillar itself usually 4-10 cmSung (2010) also reported the morphological charactersof the fruiting body of They noted that fruitingbody grew singly from the larval head and was clavate sub-lanceolate or fusiform and distinct from the stipe which wasslender glabrous and longitudinally furrowed or ridged

The hyphae of were hyaline septate and branchedwith a width of 11-152 microm

notedhyphae as ramose septate and hyaline Arora (2013)also reported that the hyphae of as aerialcottony white to creamish or yellowish septate branched andfast growing Pathania (2015) reported that in

ere

The conidia of the anamorph were formed either singly or inchains on conidiophores The conidiophores were observedas single or branched arising from synnemata The conidiawere hyaline dimorphic having two shapes spherical andspindle shaped measuring 19 to 57 microm in diameter and 25 to63 microm in length and 053 to 241 microm in width Shin(2004) also got matching observations and reported that theimperfect forms of produced spherical orspindle shaped conidia on phialides Shreshta (2005)reported that in the shape of the firstformed conidia was cylindrical or clavate followed byglobose and ellipsoidal

Perithecia were peripheral to slightly immersed globular tooval in shape narrow towards the top with an ostioles at thetip The length of the perithecia was 826-396 microm and thewidth was 936-17127 microm Numerous elongated ascicontaining filiform ascospores which were four-partite andeight in numbers were observed within the perithecia Thesize of the ascus was about 1154-17011 times 52-10 microm and theascospore was 1053-1357 times 251-273 microm Characters of theperithecium in reported by many scientistsare in accordance with the above observations Gwangpo(2000) reported the perithecium of as either oval-shaped or egg-shaped consisting of numerous numbers ofthin long asci containing ascospores Shin (2004) alsoobserved that the apex of the stroma contains numerousperithecia with several ascospores inside asci The ascosporeswere characterized by a thread-like structure in the middlewith non disarticulating part spores attached on both sidesSung (2007) observed perithecia of assuperficial to completely immersed ordinal or oblique inarrangement Asci were hyaline cylindrical and ascosporeswere cylindrical multiseptate disarticulating into part-spores or non-disarticulating Observations of Xie(2010) and Zhong (2010) were also similar to the above

study where they found thread like ascospores in ascus Sung(2010) while working with

observed that the perithecia were ovoid in shape and semi-immersed on the stroma and the ascospores were unevenlyseptate but do not disarticulate into part-spores

The fungus was isolated on PDA medium from the fruitingbodies collected during the survey This showed that thePotato Dextrose Agar which is the most widely used mediumfor fungal isolation can be used for isolation of

Isolation in PDA medium was alreadyreported by many scientists like Sung

Molecular identification of the isolate was done bycomparison of ITS sequences at species level Sequenceanalysis of the culture showed homology with

strain KC1 having 98identity strain KC1 was reported from TamilNadu by Chinnusamy and Krishnamoorthi in 2017 (NCBIGenBank httpncbinlmnhmgovblast) Hence the presentstudy was the first report of from Kerala

The fungus was isolated from stroma stipe and sclerotia offungal-coconut root grub complex on PDA media followedby periodic sub culturing and maintenance of the isolate inPDA slants under refrigerated condition for further studiesDetailed morphological characterization of the isolate wasdone The isolate was identified at species level as

Further studies have to becarried out to unravel the medicinal properties of this fungusand there should be investigations on anti-fungal anti-bacterial and biocontrol properties along with detailed studieson mass production of this precious fungus

Authors are grateful to Kerala Agricultural UniversityThrissur Kerala India for providing facilities to carry out thisstudy in the Department of Plant Pathology at College ofHorticulture Vellanikkara and College of AgriculturePadannakkad

Arora R K 2015 (Berk) Sacc- anentomophagous medicinal fungus-a review

0161-0170

Arora R K Singh N and Singh R P 2013Characterization of an entomophagous medicinalfungus (Berk) Sacc ofUttarakhand India 195-200

Chinnusamy S and Krishnamoorthy A S 2017Identification of 3deoxyadenosine (Cordycepin)

O sinensiset al

et alO sinensis

Cordyceps

et alCordyceps sinensis

et alCordyceps militaris

viz

et al

Ophiocordycepset al

Cordyceps militaris

Ophiocordyceps

C sinensis

et al

et al Ophiocordyceps

et alet al

et al Cordyceps cardinalis

Ophiocordyceps

Ophiocordyceps neovolkianaO neovolkiana

O neovolkiana

Ophiocordyceps neovolkiana

Cordyceps sinensisInt J

Adv Multidiscip Res

Cordyceps sinensis The bioscan

the hyphae w thin-walled branchedaround148 - 161μmbroad

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

2

8

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et alC sinensis

et alet al

59P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

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mycelium from caterpillarfungus fruiting body (1)1-21

Kobayasi Y and Shimizu D 1980 species fromJapan 2

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Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub

Burm(3A) 614-618

Li Y Wang X L Jiao L Jiang Y Li H Jiang S PLhosumtseiring N Fu S Z Dong C H Zhan Yand Yao YJ 2011 A survey of the geographicdistribution of

(6) 913-919

Nam Sung-Hee Li Chun-Ru Hong In-Pyo Sung Kyu-Byoung Kang Seok-Woo Fan Mei-Zhen Li andZeng-Zhi 2006 Isolation and Culture ofE n t om opa t hog en i c Fun gus

(2) 57-61

Pathania P Joshi M and Sagar A 2015 MorphologicalPhysiological and molecular studies on wildlycollected from North WestHlmalayas India (1)53-62

Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146

Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

Shin J C Shrestha B Lee W H Park Y J Kim S YJeong G R Kim H K Kim T W and Sung J M2004 Distribution and favorable conditions formycelial growth of in Korea

(2) 79-88

Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in

(2) 69-76

Shrestha B Weimin Z Yongjie Z and Xingzhong L2010 What is the Chinese caterpillar fungus

( ) (4) 228-236

Sridhar K R and Karun N C 2017 Observations onin the Western Ghats

(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

Xie F Zhu Z X Wei K L Zhang S X Zhang N andTian W 2010 Effects of different culture media onthe hyphal growth of strains

(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

Ophiocordyceps sinensisJ Microbiol Methods

5

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60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Page 2: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

carried out Various characters like shape size and colour ofsclerotia stipe and stroma and branching pattern of the stromaassociated with the insect-fungal complex were studied andobservations were recorded

Microscopic slides were prepared using lactophenol cottonblue stain and observed under compound microscope at 5x10x 40x and 100x magnifications to study the anamorphicstage including hyphae synnemata conidiophores andconidia and teleomorphic stage including perithecia asciand ascospores of the fungus Measurements andphotomicrographs were taken using Zeiss Axiolab imageanalyzer

Molecular characterization of the isolate was done to identifythe isolate at species level by DNA barcoding using universalprimers of ITS sequences Sequence analysis and nucleotidehomology of the fungus was analysed through the BLASTprogramme of NCBI (http ncbinlmnhmgovblast)Sequence analysis of the culture showed homology with

having 98 per cent identity Thesequence of the fungus was deposited at the GenBank of NCBIwithaccessionnumberMH668282and theculturewasdepositedin National Fungal Culture Collection of India at MACSAgharkarInstitutePunewithculturenumberNFCCI4331

Purposive sampling surveys were conducted in threelocations of coastal sandy tracts of Kasargod district duringJune to September months of 2017 and 2018 The fruitingbodies of sp emerged from the parasitized grubwere collected from three locations Instructional Farm ofCollege of Agriculture Padannakkad Valiyaparamba area ofNileshwar and Regional Agricultural Research StationPilicode where the coconut root grub is an endemicpolyphagous pest

The fruit bodies were found to be emerged from the soil aftersummer showers usually during mid May and after the onsetof monsoon showers during early June The population offruit bodies in one square meter area of soil were assessed inall the three locations and represented in the Amongthe three areas surveyed maximum number of fruiting bodies(average of 1386 m ) were found at Instructional Farm ofCollege ofAgriculture Padannakkad Kasargod

The fungus was isolated on PDAmedium and namedasCD1( )andwas brought intopurecultureThemyceliaappeared initially white later turning to creamish white tosalmon colour ( ) and the underside of the plate beinglight brown ( ) Subsequently numerous thread likepinkish synnemata appeared in the culture representing theanamorphic stage sp The anamorph producedsimilar type ofconidiaas produced in thefield

The fruit body of the fungus wasfound to be emerging from the cephalic region of the body ofthird instar grub of The club-shaped upper portion of fruit body called as stroma which isthe fertile portion in which the perithecia were present themiddle stalk-like part stipe and the mummified dead grubknown as sclerotium were observed ( ) The stipe andstroma portion of the fruiting bodies were found to be abovethe soil and infected grub was located at a depth of around 5 to10 cm below the soil ( )Average length and girth of thefruiting bodies obtained from all the three locations weremeasured ( ) in which the maximum length of 119 cmand 16 cm for stipe and stroma respectively were obtainedfrom Instructional farm of College of AgriculturePadannakkad Maximum girth of 39 cm and 17 cm for stipeand stroma was also observed from the same soil ( )The longitudinal section of the fruiting body showedcomplete mycelial colonization of the insect body by thefungus where the internal organs of the grub were replacedwith fungal mycelium ( )

Comprehensive study on the process of fruit body emergencewas carried out in all the three locations From the buriedinfected grubs yellowish-orange coloured stalks of about03-06 cm thickness emerged during the last week of May orfirst week of June and the tip of which later became bulgedand turned to bright orange red in colour Stromatal headswere observed to emerge from the cephalic region of theinfected third instar grubs of After 40 days bythe second week of July the stroma region showed numeroushumps which were initially orange red in colour turning tobrown representing formation of perithecia The colourturned to dark-brown with the complete maturation ofperithecia and the release of ascospores occurred in a periodof two weeks During August after an interval of two weeksthe stroma region became dark-brown to black and numerouswhite colored hook like sporulating structures calledsynnemata were developed representing the conidial stage of

Ophiocordyceps neovolkiana

Cordycepsviz

Hirsutella

Leucopholis coneophora

L coneophora

RESULTSSurvey

Table 1

IsolationFig 1a

Fig 1bFig 1c

Morphological studies

Fig 1d

Fig 1e

Table 2

Fig 1f

Fig 1g2

Number of fruiting bodies m2Mean2017 2018

Padannakkad 1525 1250 1386Valiaparamba 575 450 513

Pilicode 275 225 250

Table 1 Extent of fruit bodies in three locations (mean of fourreplications)

Fig1aFig1bampcFig1dFig1eFig1fFig1g

Typical colonies of sp on isolation plateCultureplate of spAssociationof spwith

sp in naturalhabitatVariationin sizeof fruitbodyL Sof sp

OphiocordycepsOphiocordyceps

Ophiocordyceps Leucopholis coneophoraOphiocordyceps

Ophiocordyceps

56 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Fig 3aFig 3bFig 3c

Fig 3dFig 3eFig 3fFig 3g

Hypha of spSynnemataDifferent stages of development of synnemata on thefruiting body in natural habitatSingle spherical conidia on conidiophoresSpherical conidiaSpindle conidia in chain on conidiophoresSpindle conidia

Hirsutella

Description Dimension (microm)

Hypha Hyaline septate and branched 11- 152

Synnemata Cylidrical with numerous conidiophores 2136 -2351 times 991-1100

Conidiophore Elongated single or branched 564-878

Spherical conidia Born singly on conidiophore 19-57

Spindle conidia Born in chains on conidiophore 25-63 times 053-241

Table 3 Microscopic characters of anamorphic stage

the fungus ( sp) ( ) Hence a gradual changeof colour and texture of stroma was seen during differentstages of development ( ) Branching of stroma wasnoticed as double and very rarely three to five branches from asingle stroma were observed ( ) After one month bythe second week of September the whole fruiting body gotdecomposed by the action of soil dwelling saprophytesVarious other macroscopic characters like shape of the fruitbody colour of sclerotium stipe and stroma collected fromthe three locations surveyed were also studied and theobservations are shown in

Microscopic observations ofsp such as characters of anamorphic stage

including hyphae synnemata conidiophores and conidia andteleomorphic stage including perithecia asci and ascosporeswere taken

Hyphae were hyaline septate and branched with a width of 11to 152 microm ( ) The length and width of synnematafrom which the conidiophores were arising was found to be2136 to 2351 microm and 991 to 1100 microm respectively ( )Synnemata was found to have cylindrical shape withnumerous conidiophores all over the surface In naturalhabitat the synnemata started to form initially as whitecottony cushion later turning to light pinkish white hook likestructures on the stroma having a length of 02-13 cm long( )

The conidia were formed either singly or in chains on longconidiophores measuring 564 to 878 microm Theconidiophores can be single or branched The conidia weredimorphic and hyaline among which one type of the conidia

was spherical with a diameter of 19 to 57 microm ( ) andthe other was spindle shaped with a length of 25 to 63 micromand width of 053 to 241 microm ( ) The sphericalconidia were arranged singly ( ) and spindle shapedconidia were arranged in chains on the conidiophores (

)

Microscopic examination of thin cross sections of stromashowed numerous perithecia towards the peripheral regions( ) They were peripheral to slightly immersedglobular to oval in shape narrow towards the tip ( )Openings of perithecia the ostioles through which releaseof ascospores to the outside occurs were found at the tip( ) The length and width of perithecia was found to be826 to 3961 microm and 936 to 1713 microm respectively Thethickness of the perithecial wall ranged from 166 to 217microm ( ) On the examination of perithecianumerous elongated asci were observed inside theperithecium Crozier formation stage during the ascusdevelopment was observed in immature perithecium (

) On maturation asci were observed as a bundle insidethe perithecium ( ) The ascus was elongated withlength ranging from 1154 to 1701 microm and width of 52 to10 microm ( ) The tip of the ascus had a prominent cap oroperculum the lid-like structure through which theascospores are released out of ascus ( ) The ascuspossesses eight filiform ascospores ( ) which werefour-partite with 1053 to 1357 microm length and 25 microm to 27microm width ( ) The ascospores usually split into four

Hirsutella

Ophiocordyceps

Fig 2a

Fig 2b

Fig 2c

Table 2

Microscopic studies

Fig 3a

Fig 3b

Fig 3c

Fig 3e

Fig 3gFig 3d

Fig3f Table 3

Fig 4aFig 4b

Fig 4c

Figs 4def g

Fig5a

Fig 5b

Fig 5c

Fig 5dFig 5e

Fig 5f

Characters of fruiting bodyShape Size (cm) Colour

Length Girth Sclerotia Stipe SromaStipe Stroma Stipe Stroma

Padannakkad Spindle 119 39 16 17 Black Dark-brown

Bright orange turningto dark-brown

Valiaparamba Club 85 28 13 14 Dark-brown

Dark-brown

Light-brown turningto dark-brown

Pilicode Club 104 32 12 14 Dark-brown

Brown Reddish-brownturning to dark-brown

Table 2 Macroscopic characters of sp in three locations(mean of 15 replicates)

Cordyceps

Fig 2aFig 2bFig 2c

Different stages of fruiting body development in the fieldChanges in stroma during developmentBranching pattern of stroma

57P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

Fig 5aFig 5bFig 5cFig 5dFig 5eFig 5fFig 5g

Crozier formation during ascus developmentMass of mature asciAsciAscus with operculumEight ascospores in an ascusAscosporesPart spores of ascospore

Description Diamension (microm)

Perithecia Globular -oval with ostiole 826-3961 times 936-17127

Perithecial Wall Thick 166-2165

Ascus Elongated 1154-17011 times 52-10

Acsospore Filiform and four partite 1053-1357 times 251-273

Ascospore partspore

Terminal spore pointed at the tip and middlespores are cylindrical

2558-4459 times 252-274

Table 4 Microscopic characters of teleomorphic stage

part spores in which the middle two spores werecylindrical and the terminal spores were pointed at the tiphaving 2558 to 4459 microm length and 252-274 microm width( )

a well-known and valued traditionalChinese medicinal mushroom also called Dong Chong XiaCao (winter worm summer grass) is a parasitic complex offungus and caterpillar of family

Due to the presence of many bioactive compoundslike adenosine cordycepin ergosterol havebeen used as food as well as in herbal medicine to treatrespiratory pulmonary renal liver and cardiovasculardiseases hyposexuality and hyperlipidemia (Arora 2015)Even though the distribution of this fungus is cosmopolitanmany of its members like are mostly confined tohigh Himalayan Mountains in China Tibet Nepal Bhutan andIndia at an altitude of 3000 to 5000 msl (Sharma 2004) Butfrom the surveys conducted in costal sandy tracts of KasargodDistrict of Kerala it was found that spparasitizes the third instar grubs of The present observations are in accordance with the report of(Kumar and Aparna 2014) Sreedhar and Karun (2017)reported the presence of associatedwith sap-sucking pentatomid stinkbug inWestern Ghats Dattaraj (2018) documented reports byvarious scientists about the occurrence of and alliedspecies in the Western Ghats and southwest coast of India andalso reported six species of recovered in the scrubjunglesof southwest Karnataka

So far there are no systematic studies conducted on themycological aspects of sp from Kerala andtherefore this study is a first attempt for the isolation andmorphological characterization of this fungus The period ofoccurrence of sp as per the samplingsurveys conducted in three selected locations of Kasargoddistrict during 2017 and 2018 has similarity with reports ofother scientists in different countries It was reported that thebest time of collection ofspecimens is from July to September in Japan (Kobayasi andShimizu 1980) and Korea (Nam 2006) The presence ofthe fungus in tropical conditions at low altitudes of Keralamay be attributed to the high humidity and low temperaturesituations prevailing in the state during south-west monsoonperiods the time at which the fungus was noticed in the field(Kumar andAparna 2014)

In the fruiting bodies of sp the bulged tipportion was observed as stroma and stalk like structure asstipe and sclerotium was the mummified grub which was

Fig 5g Table 4DISCUSSIONOphiocordyceps sinensis

Hepialis armoricanusHepialidae

Ophiocordyceps

O sinensis

OphiocordycepsLeucopholis coneophora

Ophiocordyceps nutansHalyomorpha halys

et alCordyceps

Cordyceps

Ophiocordyceps

Ophiocordyceps

Cordyceps sphecocephala

et al

Ophiocordyceps

Fig4aFig 4bFig 4cFig 4dFig 4eFig 4fFig 4g

StromaPeritheciaOstioleC S of stromaPeripheral arrangement of peritheciaGlobular to oval peritheciaMicroscopic characters of stroma

58 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

completely filled with mycelia of the fungus The tips ofperithecia were found as numerous humps which are initiallyorange red in colour turning to dark-brown These findingswere having similarity with observations of other researchersThe morphological characters of was described byShrestha (2010) as it consists of two parts a fungalendosclerotium (caterpillar) and stroma which was initiallyyellow in colour and later turning to dark brown or blackwhich was longer than the caterpillar itself usually 4-10 cmSung (2010) also reported the morphological charactersof the fruiting body of They noted that fruitingbody grew singly from the larval head and was clavate sub-lanceolate or fusiform and distinct from the stipe which wasslender glabrous and longitudinally furrowed or ridged

The hyphae of were hyaline septate and branchedwith a width of 11-152 microm

notedhyphae as ramose septate and hyaline Arora (2013)also reported that the hyphae of as aerialcottony white to creamish or yellowish septate branched andfast growing Pathania (2015) reported that in

ere

The conidia of the anamorph were formed either singly or inchains on conidiophores The conidiophores were observedas single or branched arising from synnemata The conidiawere hyaline dimorphic having two shapes spherical andspindle shaped measuring 19 to 57 microm in diameter and 25 to63 microm in length and 053 to 241 microm in width Shin(2004) also got matching observations and reported that theimperfect forms of produced spherical orspindle shaped conidia on phialides Shreshta (2005)reported that in the shape of the firstformed conidia was cylindrical or clavate followed byglobose and ellipsoidal

Perithecia were peripheral to slightly immersed globular tooval in shape narrow towards the top with an ostioles at thetip The length of the perithecia was 826-396 microm and thewidth was 936-17127 microm Numerous elongated ascicontaining filiform ascospores which were four-partite andeight in numbers were observed within the perithecia Thesize of the ascus was about 1154-17011 times 52-10 microm and theascospore was 1053-1357 times 251-273 microm Characters of theperithecium in reported by many scientistsare in accordance with the above observations Gwangpo(2000) reported the perithecium of as either oval-shaped or egg-shaped consisting of numerous numbers ofthin long asci containing ascospores Shin (2004) alsoobserved that the apex of the stroma contains numerousperithecia with several ascospores inside asci The ascosporeswere characterized by a thread-like structure in the middlewith non disarticulating part spores attached on both sidesSung (2007) observed perithecia of assuperficial to completely immersed ordinal or oblique inarrangement Asci were hyaline cylindrical and ascosporeswere cylindrical multiseptate disarticulating into part-spores or non-disarticulating Observations of Xie(2010) and Zhong (2010) were also similar to the above

study where they found thread like ascospores in ascus Sung(2010) while working with

observed that the perithecia were ovoid in shape and semi-immersed on the stroma and the ascospores were unevenlyseptate but do not disarticulate into part-spores

The fungus was isolated on PDA medium from the fruitingbodies collected during the survey This showed that thePotato Dextrose Agar which is the most widely used mediumfor fungal isolation can be used for isolation of

Isolation in PDA medium was alreadyreported by many scientists like Sung

Molecular identification of the isolate was done bycomparison of ITS sequences at species level Sequenceanalysis of the culture showed homology with

strain KC1 having 98identity strain KC1 was reported from TamilNadu by Chinnusamy and Krishnamoorthi in 2017 (NCBIGenBank httpncbinlmnhmgovblast) Hence the presentstudy was the first report of from Kerala

The fungus was isolated from stroma stipe and sclerotia offungal-coconut root grub complex on PDA media followedby periodic sub culturing and maintenance of the isolate inPDA slants under refrigerated condition for further studiesDetailed morphological characterization of the isolate wasdone The isolate was identified at species level as

Further studies have to becarried out to unravel the medicinal properties of this fungusand there should be investigations on anti-fungal anti-bacterial and biocontrol properties along with detailed studieson mass production of this precious fungus

Authors are grateful to Kerala Agricultural UniversityThrissur Kerala India for providing facilities to carry out thisstudy in the Department of Plant Pathology at College ofHorticulture Vellanikkara and College of AgriculturePadannakkad

Arora R K 2015 (Berk) Sacc- anentomophagous medicinal fungus-a review

0161-0170

Arora R K Singh N and Singh R P 2013Characterization of an entomophagous medicinalfungus (Berk) Sacc ofUttarakhand India 195-200

Chinnusamy S and Krishnamoorthy A S 2017Identification of 3deoxyadenosine (Cordycepin)

O sinensiset al

et alO sinensis

Cordyceps

et alCordyceps sinensis

et alCordyceps militaris

viz

et al

Ophiocordycepset al

Cordyceps militaris

Ophiocordyceps

C sinensis

et al

et al Ophiocordyceps

et alet al

et al Cordyceps cardinalis

Ophiocordyceps

Ophiocordyceps neovolkianaO neovolkiana

O neovolkiana

Ophiocordyceps neovolkiana

Cordyceps sinensisInt J

Adv Multidiscip Res

Cordyceps sinensis The bioscan

the hyphae w thin-walled branchedaround148 - 161μmbroad

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

2

8

Chen J Zang W Lu T Zeng Y and Kong L 2006Morphological and genetic characterization of ac u l t i v a t e d fu n gu s a n d i t spolysaccharaide component possessing antioxidantproperty in H22 tumor- bearing mice 2742-2748

Co rd y c e ps

Life Sci 78

Chen (2006) observedsimilar characters for the hyphae of They

(2011) SangdeeandSangdee(2013)and Ko (2017)

et alC sinensis

et alet al

59P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

from the medicinal mushroomsspp (3) 788-792

Dattaraj H R Jagadish B R Sridhar K R and Ghate SD 2018 Are the scrub jungles of Southwest Indiapotential habitats of 20-22

Gwangpo K 2000 Characteristics of species(5) 537-539

Holliday J and Cleaver M 2004 On the Trail of the YakAncient in the modern world

Ko Y F Liau J C Lee C S Chiu C Y Martel J Lin CS Tseng S F Ojcius D M Lai H C and YoungJ D 2017 Isolation culture and characterization of

mycelium from caterpillarfungus fruiting body (1)1-21

Kobayasi Y and Shimizu D 1980 species fromJapan 2

77-96

Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub

Burm(3A) 614-618

Li Y Wang X L Jiao L Jiang Y Li H Jiang S PLhosumtseiring N Fu S Z Dong C H Zhan Yand Yao YJ 2011 A survey of the geographicdistribution of

(6) 913-919

Nam Sung-Hee Li Chun-Ru Hong In-Pyo Sung Kyu-Byoung Kang Seok-Woo Fan Mei-Zhen Li andZeng-Zhi 2006 Isolation and Culture ofE n t om opa t hog en i c Fun gus

(2) 57-61

Pathania P Joshi M and Sagar A 2015 MorphologicalPhysiological and molecular studies on wildlycollected from North WestHlmalayas India (1)53-62

Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146

Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

Shin J C Shrestha B Lee W H Park Y J Kim S YJeong G R Kim H K Kim T W and Sung J M2004 Distribution and favorable conditions formycelial growth of in Korea

(2) 79-88

Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in

(2) 69-76

Shrestha B Weimin Z Yongjie Z and Xingzhong L2010 What is the Chinese caterpillar fungus

( ) (4) 228-236

Sridhar K R and Karun N C 2017 Observations onin the Western Ghats

(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

Xie F Zhu Z X Wei K L Zhang S X Zhang N andTian W 2010 Effects of different culture media onthe hyphal growth of strains

(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

Ophiocordyceps sinensisJ Microbiol Methods

5

51

11

12

8

49

13

3

7

86

32

33

1

6

57

39

38

22

83

60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Page 3: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

Fig 3aFig 3bFig 3c

Fig 3dFig 3eFig 3fFig 3g

Hypha of spSynnemataDifferent stages of development of synnemata on thefruiting body in natural habitatSingle spherical conidia on conidiophoresSpherical conidiaSpindle conidia in chain on conidiophoresSpindle conidia

Hirsutella

Description Dimension (microm)

Hypha Hyaline septate and branched 11- 152

Synnemata Cylidrical with numerous conidiophores 2136 -2351 times 991-1100

Conidiophore Elongated single or branched 564-878

Spherical conidia Born singly on conidiophore 19-57

Spindle conidia Born in chains on conidiophore 25-63 times 053-241

Table 3 Microscopic characters of anamorphic stage

the fungus ( sp) ( ) Hence a gradual changeof colour and texture of stroma was seen during differentstages of development ( ) Branching of stroma wasnoticed as double and very rarely three to five branches from asingle stroma were observed ( ) After one month bythe second week of September the whole fruiting body gotdecomposed by the action of soil dwelling saprophytesVarious other macroscopic characters like shape of the fruitbody colour of sclerotium stipe and stroma collected fromthe three locations surveyed were also studied and theobservations are shown in

Microscopic observations ofsp such as characters of anamorphic stage

including hyphae synnemata conidiophores and conidia andteleomorphic stage including perithecia asci and ascosporeswere taken

Hyphae were hyaline septate and branched with a width of 11to 152 microm ( ) The length and width of synnematafrom which the conidiophores were arising was found to be2136 to 2351 microm and 991 to 1100 microm respectively ( )Synnemata was found to have cylindrical shape withnumerous conidiophores all over the surface In naturalhabitat the synnemata started to form initially as whitecottony cushion later turning to light pinkish white hook likestructures on the stroma having a length of 02-13 cm long( )

The conidia were formed either singly or in chains on longconidiophores measuring 564 to 878 microm Theconidiophores can be single or branched The conidia weredimorphic and hyaline among which one type of the conidia

was spherical with a diameter of 19 to 57 microm ( ) andthe other was spindle shaped with a length of 25 to 63 micromand width of 053 to 241 microm ( ) The sphericalconidia were arranged singly ( ) and spindle shapedconidia were arranged in chains on the conidiophores (

)

Microscopic examination of thin cross sections of stromashowed numerous perithecia towards the peripheral regions( ) They were peripheral to slightly immersedglobular to oval in shape narrow towards the tip ( )Openings of perithecia the ostioles through which releaseof ascospores to the outside occurs were found at the tip( ) The length and width of perithecia was found to be826 to 3961 microm and 936 to 1713 microm respectively Thethickness of the perithecial wall ranged from 166 to 217microm ( ) On the examination of perithecianumerous elongated asci were observed inside theperithecium Crozier formation stage during the ascusdevelopment was observed in immature perithecium (

) On maturation asci were observed as a bundle insidethe perithecium ( ) The ascus was elongated withlength ranging from 1154 to 1701 microm and width of 52 to10 microm ( ) The tip of the ascus had a prominent cap oroperculum the lid-like structure through which theascospores are released out of ascus ( ) The ascuspossesses eight filiform ascospores ( ) which werefour-partite with 1053 to 1357 microm length and 25 microm to 27microm width ( ) The ascospores usually split into four

Hirsutella

Ophiocordyceps

Fig 2a

Fig 2b

Fig 2c

Table 2

Microscopic studies

Fig 3a

Fig 3b

Fig 3c

Fig 3e

Fig 3gFig 3d

Fig3f Table 3

Fig 4aFig 4b

Fig 4c

Figs 4def g

Fig5a

Fig 5b

Fig 5c

Fig 5dFig 5e

Fig 5f

Characters of fruiting bodyShape Size (cm) Colour

Length Girth Sclerotia Stipe SromaStipe Stroma Stipe Stroma

Padannakkad Spindle 119 39 16 17 Black Dark-brown

Bright orange turningto dark-brown

Valiaparamba Club 85 28 13 14 Dark-brown

Dark-brown

Light-brown turningto dark-brown

Pilicode Club 104 32 12 14 Dark-brown

Brown Reddish-brownturning to dark-brown

Table 2 Macroscopic characters of sp in three locations(mean of 15 replicates)

Cordyceps

Fig 2aFig 2bFig 2c

Different stages of fruiting body development in the fieldChanges in stroma during developmentBranching pattern of stroma

57P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

Fig 5aFig 5bFig 5cFig 5dFig 5eFig 5fFig 5g

Crozier formation during ascus developmentMass of mature asciAsciAscus with operculumEight ascospores in an ascusAscosporesPart spores of ascospore

Description Diamension (microm)

Perithecia Globular -oval with ostiole 826-3961 times 936-17127

Perithecial Wall Thick 166-2165

Ascus Elongated 1154-17011 times 52-10

Acsospore Filiform and four partite 1053-1357 times 251-273

Ascospore partspore

Terminal spore pointed at the tip and middlespores are cylindrical

2558-4459 times 252-274

Table 4 Microscopic characters of teleomorphic stage

part spores in which the middle two spores werecylindrical and the terminal spores were pointed at the tiphaving 2558 to 4459 microm length and 252-274 microm width( )

a well-known and valued traditionalChinese medicinal mushroom also called Dong Chong XiaCao (winter worm summer grass) is a parasitic complex offungus and caterpillar of family

Due to the presence of many bioactive compoundslike adenosine cordycepin ergosterol havebeen used as food as well as in herbal medicine to treatrespiratory pulmonary renal liver and cardiovasculardiseases hyposexuality and hyperlipidemia (Arora 2015)Even though the distribution of this fungus is cosmopolitanmany of its members like are mostly confined tohigh Himalayan Mountains in China Tibet Nepal Bhutan andIndia at an altitude of 3000 to 5000 msl (Sharma 2004) Butfrom the surveys conducted in costal sandy tracts of KasargodDistrict of Kerala it was found that spparasitizes the third instar grubs of The present observations are in accordance with the report of(Kumar and Aparna 2014) Sreedhar and Karun (2017)reported the presence of associatedwith sap-sucking pentatomid stinkbug inWestern Ghats Dattaraj (2018) documented reports byvarious scientists about the occurrence of and alliedspecies in the Western Ghats and southwest coast of India andalso reported six species of recovered in the scrubjunglesof southwest Karnataka

So far there are no systematic studies conducted on themycological aspects of sp from Kerala andtherefore this study is a first attempt for the isolation andmorphological characterization of this fungus The period ofoccurrence of sp as per the samplingsurveys conducted in three selected locations of Kasargoddistrict during 2017 and 2018 has similarity with reports ofother scientists in different countries It was reported that thebest time of collection ofspecimens is from July to September in Japan (Kobayasi andShimizu 1980) and Korea (Nam 2006) The presence ofthe fungus in tropical conditions at low altitudes of Keralamay be attributed to the high humidity and low temperaturesituations prevailing in the state during south-west monsoonperiods the time at which the fungus was noticed in the field(Kumar andAparna 2014)

In the fruiting bodies of sp the bulged tipportion was observed as stroma and stalk like structure asstipe and sclerotium was the mummified grub which was

Fig 5g Table 4DISCUSSIONOphiocordyceps sinensis

Hepialis armoricanusHepialidae

Ophiocordyceps

O sinensis

OphiocordycepsLeucopholis coneophora

Ophiocordyceps nutansHalyomorpha halys

et alCordyceps

Cordyceps

Ophiocordyceps

Ophiocordyceps

Cordyceps sphecocephala

et al

Ophiocordyceps

Fig4aFig 4bFig 4cFig 4dFig 4eFig 4fFig 4g

StromaPeritheciaOstioleC S of stromaPeripheral arrangement of peritheciaGlobular to oval peritheciaMicroscopic characters of stroma

58 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

completely filled with mycelia of the fungus The tips ofperithecia were found as numerous humps which are initiallyorange red in colour turning to dark-brown These findingswere having similarity with observations of other researchersThe morphological characters of was described byShrestha (2010) as it consists of two parts a fungalendosclerotium (caterpillar) and stroma which was initiallyyellow in colour and later turning to dark brown or blackwhich was longer than the caterpillar itself usually 4-10 cmSung (2010) also reported the morphological charactersof the fruiting body of They noted that fruitingbody grew singly from the larval head and was clavate sub-lanceolate or fusiform and distinct from the stipe which wasslender glabrous and longitudinally furrowed or ridged

The hyphae of were hyaline septate and branchedwith a width of 11-152 microm

notedhyphae as ramose septate and hyaline Arora (2013)also reported that the hyphae of as aerialcottony white to creamish or yellowish septate branched andfast growing Pathania (2015) reported that in

ere

The conidia of the anamorph were formed either singly or inchains on conidiophores The conidiophores were observedas single or branched arising from synnemata The conidiawere hyaline dimorphic having two shapes spherical andspindle shaped measuring 19 to 57 microm in diameter and 25 to63 microm in length and 053 to 241 microm in width Shin(2004) also got matching observations and reported that theimperfect forms of produced spherical orspindle shaped conidia on phialides Shreshta (2005)reported that in the shape of the firstformed conidia was cylindrical or clavate followed byglobose and ellipsoidal

Perithecia were peripheral to slightly immersed globular tooval in shape narrow towards the top with an ostioles at thetip The length of the perithecia was 826-396 microm and thewidth was 936-17127 microm Numerous elongated ascicontaining filiform ascospores which were four-partite andeight in numbers were observed within the perithecia Thesize of the ascus was about 1154-17011 times 52-10 microm and theascospore was 1053-1357 times 251-273 microm Characters of theperithecium in reported by many scientistsare in accordance with the above observations Gwangpo(2000) reported the perithecium of as either oval-shaped or egg-shaped consisting of numerous numbers ofthin long asci containing ascospores Shin (2004) alsoobserved that the apex of the stroma contains numerousperithecia with several ascospores inside asci The ascosporeswere characterized by a thread-like structure in the middlewith non disarticulating part spores attached on both sidesSung (2007) observed perithecia of assuperficial to completely immersed ordinal or oblique inarrangement Asci were hyaline cylindrical and ascosporeswere cylindrical multiseptate disarticulating into part-spores or non-disarticulating Observations of Xie(2010) and Zhong (2010) were also similar to the above

study where they found thread like ascospores in ascus Sung(2010) while working with

observed that the perithecia were ovoid in shape and semi-immersed on the stroma and the ascospores were unevenlyseptate but do not disarticulate into part-spores

The fungus was isolated on PDA medium from the fruitingbodies collected during the survey This showed that thePotato Dextrose Agar which is the most widely used mediumfor fungal isolation can be used for isolation of

Isolation in PDA medium was alreadyreported by many scientists like Sung

Molecular identification of the isolate was done bycomparison of ITS sequences at species level Sequenceanalysis of the culture showed homology with

strain KC1 having 98identity strain KC1 was reported from TamilNadu by Chinnusamy and Krishnamoorthi in 2017 (NCBIGenBank httpncbinlmnhmgovblast) Hence the presentstudy was the first report of from Kerala

The fungus was isolated from stroma stipe and sclerotia offungal-coconut root grub complex on PDA media followedby periodic sub culturing and maintenance of the isolate inPDA slants under refrigerated condition for further studiesDetailed morphological characterization of the isolate wasdone The isolate was identified at species level as

Further studies have to becarried out to unravel the medicinal properties of this fungusand there should be investigations on anti-fungal anti-bacterial and biocontrol properties along with detailed studieson mass production of this precious fungus

Authors are grateful to Kerala Agricultural UniversityThrissur Kerala India for providing facilities to carry out thisstudy in the Department of Plant Pathology at College ofHorticulture Vellanikkara and College of AgriculturePadannakkad

Arora R K 2015 (Berk) Sacc- anentomophagous medicinal fungus-a review

0161-0170

Arora R K Singh N and Singh R P 2013Characterization of an entomophagous medicinalfungus (Berk) Sacc ofUttarakhand India 195-200

Chinnusamy S and Krishnamoorthy A S 2017Identification of 3deoxyadenosine (Cordycepin)

O sinensiset al

et alO sinensis

Cordyceps

et alCordyceps sinensis

et alCordyceps militaris

viz

et al

Ophiocordycepset al

Cordyceps militaris

Ophiocordyceps

C sinensis

et al

et al Ophiocordyceps

et alet al

et al Cordyceps cardinalis

Ophiocordyceps

Ophiocordyceps neovolkianaO neovolkiana

O neovolkiana

Ophiocordyceps neovolkiana

Cordyceps sinensisInt J

Adv Multidiscip Res

Cordyceps sinensis The bioscan

the hyphae w thin-walled branchedaround148 - 161μmbroad

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

2

8

Chen J Zang W Lu T Zeng Y and Kong L 2006Morphological and genetic characterization of ac u l t i v a t e d fu n gu s a n d i t spolysaccharaide component possessing antioxidantproperty in H22 tumor- bearing mice 2742-2748

Co rd y c e ps

Life Sci 78

Chen (2006) observedsimilar characters for the hyphae of They

(2011) SangdeeandSangdee(2013)and Ko (2017)

et alC sinensis

et alet al

59P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

from the medicinal mushroomsspp (3) 788-792

Dattaraj H R Jagadish B R Sridhar K R and Ghate SD 2018 Are the scrub jungles of Southwest Indiapotential habitats of 20-22

Gwangpo K 2000 Characteristics of species(5) 537-539

Holliday J and Cleaver M 2004 On the Trail of the YakAncient in the modern world

Ko Y F Liau J C Lee C S Chiu C Y Martel J Lin CS Tseng S F Ojcius D M Lai H C and YoungJ D 2017 Isolation culture and characterization of

mycelium from caterpillarfungus fruiting body (1)1-21

Kobayasi Y and Shimizu D 1980 species fromJapan 2

77-96

Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub

Burm(3A) 614-618

Li Y Wang X L Jiao L Jiang Y Li H Jiang S PLhosumtseiring N Fu S Z Dong C H Zhan Yand Yao YJ 2011 A survey of the geographicdistribution of

(6) 913-919

Nam Sung-Hee Li Chun-Ru Hong In-Pyo Sung Kyu-Byoung Kang Seok-Woo Fan Mei-Zhen Li andZeng-Zhi 2006 Isolation and Culture ofE n t om opa t hog en i c Fun gus

(2) 57-61

Pathania P Joshi M and Sagar A 2015 MorphologicalPhysiological and molecular studies on wildlycollected from North WestHlmalayas India (1)53-62

Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146

Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

Shin J C Shrestha B Lee W H Park Y J Kim S YJeong G R Kim H K Kim T W and Sung J M2004 Distribution and favorable conditions formycelial growth of in Korea

(2) 79-88

Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in

(2) 69-76

Shrestha B Weimin Z Yongjie Z and Xingzhong L2010 What is the Chinese caterpillar fungus

( ) (4) 228-236

Sridhar K R and Karun N C 2017 Observations onin the Western Ghats

(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

Xie F Zhu Z X Wei K L Zhang S X Zhang N andTian W 2010 Effects of different culture media onthe hyphal growth of strains

(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

Ophiocordyceps sinensisJ Microbiol Methods

5

51

11

12

8

49

13

3

7

86

32

33

1

6

57

39

38

22

83

60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Page 4: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

Fig 5aFig 5bFig 5cFig 5dFig 5eFig 5fFig 5g

Crozier formation during ascus developmentMass of mature asciAsciAscus with operculumEight ascospores in an ascusAscosporesPart spores of ascospore

Description Diamension (microm)

Perithecia Globular -oval with ostiole 826-3961 times 936-17127

Perithecial Wall Thick 166-2165

Ascus Elongated 1154-17011 times 52-10

Acsospore Filiform and four partite 1053-1357 times 251-273

Ascospore partspore

Terminal spore pointed at the tip and middlespores are cylindrical

2558-4459 times 252-274

Table 4 Microscopic characters of teleomorphic stage

part spores in which the middle two spores werecylindrical and the terminal spores were pointed at the tiphaving 2558 to 4459 microm length and 252-274 microm width( )

a well-known and valued traditionalChinese medicinal mushroom also called Dong Chong XiaCao (winter worm summer grass) is a parasitic complex offungus and caterpillar of family

Due to the presence of many bioactive compoundslike adenosine cordycepin ergosterol havebeen used as food as well as in herbal medicine to treatrespiratory pulmonary renal liver and cardiovasculardiseases hyposexuality and hyperlipidemia (Arora 2015)Even though the distribution of this fungus is cosmopolitanmany of its members like are mostly confined tohigh Himalayan Mountains in China Tibet Nepal Bhutan andIndia at an altitude of 3000 to 5000 msl (Sharma 2004) Butfrom the surveys conducted in costal sandy tracts of KasargodDistrict of Kerala it was found that spparasitizes the third instar grubs of The present observations are in accordance with the report of(Kumar and Aparna 2014) Sreedhar and Karun (2017)reported the presence of associatedwith sap-sucking pentatomid stinkbug inWestern Ghats Dattaraj (2018) documented reports byvarious scientists about the occurrence of and alliedspecies in the Western Ghats and southwest coast of India andalso reported six species of recovered in the scrubjunglesof southwest Karnataka

So far there are no systematic studies conducted on themycological aspects of sp from Kerala andtherefore this study is a first attempt for the isolation andmorphological characterization of this fungus The period ofoccurrence of sp as per the samplingsurveys conducted in three selected locations of Kasargoddistrict during 2017 and 2018 has similarity with reports ofother scientists in different countries It was reported that thebest time of collection ofspecimens is from July to September in Japan (Kobayasi andShimizu 1980) and Korea (Nam 2006) The presence ofthe fungus in tropical conditions at low altitudes of Keralamay be attributed to the high humidity and low temperaturesituations prevailing in the state during south-west monsoonperiods the time at which the fungus was noticed in the field(Kumar andAparna 2014)

In the fruiting bodies of sp the bulged tipportion was observed as stroma and stalk like structure asstipe and sclerotium was the mummified grub which was

Fig 5g Table 4DISCUSSIONOphiocordyceps sinensis

Hepialis armoricanusHepialidae

Ophiocordyceps

O sinensis

OphiocordycepsLeucopholis coneophora

Ophiocordyceps nutansHalyomorpha halys

et alCordyceps

Cordyceps

Ophiocordyceps

Ophiocordyceps

Cordyceps sphecocephala

et al

Ophiocordyceps

Fig4aFig 4bFig 4cFig 4dFig 4eFig 4fFig 4g

StromaPeritheciaOstioleC S of stromaPeripheral arrangement of peritheciaGlobular to oval peritheciaMicroscopic characters of stroma

58 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

completely filled with mycelia of the fungus The tips ofperithecia were found as numerous humps which are initiallyorange red in colour turning to dark-brown These findingswere having similarity with observations of other researchersThe morphological characters of was described byShrestha (2010) as it consists of two parts a fungalendosclerotium (caterpillar) and stroma which was initiallyyellow in colour and later turning to dark brown or blackwhich was longer than the caterpillar itself usually 4-10 cmSung (2010) also reported the morphological charactersof the fruiting body of They noted that fruitingbody grew singly from the larval head and was clavate sub-lanceolate or fusiform and distinct from the stipe which wasslender glabrous and longitudinally furrowed or ridged

The hyphae of were hyaline septate and branchedwith a width of 11-152 microm

notedhyphae as ramose septate and hyaline Arora (2013)also reported that the hyphae of as aerialcottony white to creamish or yellowish septate branched andfast growing Pathania (2015) reported that in

ere

The conidia of the anamorph were formed either singly or inchains on conidiophores The conidiophores were observedas single or branched arising from synnemata The conidiawere hyaline dimorphic having two shapes spherical andspindle shaped measuring 19 to 57 microm in diameter and 25 to63 microm in length and 053 to 241 microm in width Shin(2004) also got matching observations and reported that theimperfect forms of produced spherical orspindle shaped conidia on phialides Shreshta (2005)reported that in the shape of the firstformed conidia was cylindrical or clavate followed byglobose and ellipsoidal

Perithecia were peripheral to slightly immersed globular tooval in shape narrow towards the top with an ostioles at thetip The length of the perithecia was 826-396 microm and thewidth was 936-17127 microm Numerous elongated ascicontaining filiform ascospores which were four-partite andeight in numbers were observed within the perithecia Thesize of the ascus was about 1154-17011 times 52-10 microm and theascospore was 1053-1357 times 251-273 microm Characters of theperithecium in reported by many scientistsare in accordance with the above observations Gwangpo(2000) reported the perithecium of as either oval-shaped or egg-shaped consisting of numerous numbers ofthin long asci containing ascospores Shin (2004) alsoobserved that the apex of the stroma contains numerousperithecia with several ascospores inside asci The ascosporeswere characterized by a thread-like structure in the middlewith non disarticulating part spores attached on both sidesSung (2007) observed perithecia of assuperficial to completely immersed ordinal or oblique inarrangement Asci were hyaline cylindrical and ascosporeswere cylindrical multiseptate disarticulating into part-spores or non-disarticulating Observations of Xie(2010) and Zhong (2010) were also similar to the above

study where they found thread like ascospores in ascus Sung(2010) while working with

observed that the perithecia were ovoid in shape and semi-immersed on the stroma and the ascospores were unevenlyseptate but do not disarticulate into part-spores

The fungus was isolated on PDA medium from the fruitingbodies collected during the survey This showed that thePotato Dextrose Agar which is the most widely used mediumfor fungal isolation can be used for isolation of

Isolation in PDA medium was alreadyreported by many scientists like Sung

Molecular identification of the isolate was done bycomparison of ITS sequences at species level Sequenceanalysis of the culture showed homology with

strain KC1 having 98identity strain KC1 was reported from TamilNadu by Chinnusamy and Krishnamoorthi in 2017 (NCBIGenBank httpncbinlmnhmgovblast) Hence the presentstudy was the first report of from Kerala

The fungus was isolated from stroma stipe and sclerotia offungal-coconut root grub complex on PDA media followedby periodic sub culturing and maintenance of the isolate inPDA slants under refrigerated condition for further studiesDetailed morphological characterization of the isolate wasdone The isolate was identified at species level as

Further studies have to becarried out to unravel the medicinal properties of this fungusand there should be investigations on anti-fungal anti-bacterial and biocontrol properties along with detailed studieson mass production of this precious fungus

Authors are grateful to Kerala Agricultural UniversityThrissur Kerala India for providing facilities to carry out thisstudy in the Department of Plant Pathology at College ofHorticulture Vellanikkara and College of AgriculturePadannakkad

Arora R K 2015 (Berk) Sacc- anentomophagous medicinal fungus-a review

0161-0170

Arora R K Singh N and Singh R P 2013Characterization of an entomophagous medicinalfungus (Berk) Sacc ofUttarakhand India 195-200

Chinnusamy S and Krishnamoorthy A S 2017Identification of 3deoxyadenosine (Cordycepin)

O sinensiset al

et alO sinensis

Cordyceps

et alCordyceps sinensis

et alCordyceps militaris

viz

et al

Ophiocordycepset al

Cordyceps militaris

Ophiocordyceps

C sinensis

et al

et al Ophiocordyceps

et alet al

et al Cordyceps cardinalis

Ophiocordyceps

Ophiocordyceps neovolkianaO neovolkiana

O neovolkiana

Ophiocordyceps neovolkiana

Cordyceps sinensisInt J

Adv Multidiscip Res

Cordyceps sinensis The bioscan

the hyphae w thin-walled branchedaround148 - 161μmbroad

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

2

8

Chen J Zang W Lu T Zeng Y and Kong L 2006Morphological and genetic characterization of ac u l t i v a t e d fu n gu s a n d i t spolysaccharaide component possessing antioxidantproperty in H22 tumor- bearing mice 2742-2748

Co rd y c e ps

Life Sci 78

Chen (2006) observedsimilar characters for the hyphae of They

(2011) SangdeeandSangdee(2013)and Ko (2017)

et alC sinensis

et alet al

59P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi

from the medicinal mushroomsspp (3) 788-792

Dattaraj H R Jagadish B R Sridhar K R and Ghate SD 2018 Are the scrub jungles of Southwest Indiapotential habitats of 20-22

Gwangpo K 2000 Characteristics of species(5) 537-539

Holliday J and Cleaver M 2004 On the Trail of the YakAncient in the modern world

Ko Y F Liau J C Lee C S Chiu C Y Martel J Lin CS Tseng S F Ojcius D M Lai H C and YoungJ D 2017 Isolation culture and characterization of

mycelium from caterpillarfungus fruiting body (1)1-21

Kobayasi Y and Shimizu D 1980 species fromJapan 2

77-96

Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub

Burm(3A) 614-618

Li Y Wang X L Jiao L Jiang Y Li H Jiang S PLhosumtseiring N Fu S Z Dong C H Zhan Yand Yao YJ 2011 A survey of the geographicdistribution of

(6) 913-919

Nam Sung-Hee Li Chun-Ru Hong In-Pyo Sung Kyu-Byoung Kang Seok-Woo Fan Mei-Zhen Li andZeng-Zhi 2006 Isolation and Culture ofE n t om opa t hog en i c Fun gus

(2) 57-61

Pathania P Joshi M and Sagar A 2015 MorphologicalPhysiological and molecular studies on wildlycollected from North WestHlmalayas India (1)53-62

Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146

Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

Shin J C Shrestha B Lee W H Park Y J Kim S YJeong G R Kim H K Kim T W and Sung J M2004 Distribution and favorable conditions formycelial growth of in Korea

(2) 79-88

Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in

(2) 69-76

Shrestha B Weimin Z Yongjie Z and Xingzhong L2010 What is the Chinese caterpillar fungus

( ) (4) 228-236

Sridhar K R and Karun N C 2017 Observations onin the Western Ghats

(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

Xie F Zhu Z X Wei K L Zhang S X Zhang N andTian W 2010 Effects of different culture media onthe hyphal growth of strains

(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

Ophiocordyceps sinensisJ Microbiol Methods

5

51

11

12

8

49

13

3

7

86

32

33

1

6

57

39

38

22

83

60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Page 5: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

completely filled with mycelia of the fungus The tips ofperithecia were found as numerous humps which are initiallyorange red in colour turning to dark-brown These findingswere having similarity with observations of other researchersThe morphological characters of was described byShrestha (2010) as it consists of two parts a fungalendosclerotium (caterpillar) and stroma which was initiallyyellow in colour and later turning to dark brown or blackwhich was longer than the caterpillar itself usually 4-10 cmSung (2010) also reported the morphological charactersof the fruiting body of They noted that fruitingbody grew singly from the larval head and was clavate sub-lanceolate or fusiform and distinct from the stipe which wasslender glabrous and longitudinally furrowed or ridged

The hyphae of were hyaline septate and branchedwith a width of 11-152 microm

notedhyphae as ramose septate and hyaline Arora (2013)also reported that the hyphae of as aerialcottony white to creamish or yellowish septate branched andfast growing Pathania (2015) reported that in

ere

The conidia of the anamorph were formed either singly or inchains on conidiophores The conidiophores were observedas single or branched arising from synnemata The conidiawere hyaline dimorphic having two shapes spherical andspindle shaped measuring 19 to 57 microm in diameter and 25 to63 microm in length and 053 to 241 microm in width Shin(2004) also got matching observations and reported that theimperfect forms of produced spherical orspindle shaped conidia on phialides Shreshta (2005)reported that in the shape of the firstformed conidia was cylindrical or clavate followed byglobose and ellipsoidal

Perithecia were peripheral to slightly immersed globular tooval in shape narrow towards the top with an ostioles at thetip The length of the perithecia was 826-396 microm and thewidth was 936-17127 microm Numerous elongated ascicontaining filiform ascospores which were four-partite andeight in numbers were observed within the perithecia Thesize of the ascus was about 1154-17011 times 52-10 microm and theascospore was 1053-1357 times 251-273 microm Characters of theperithecium in reported by many scientistsare in accordance with the above observations Gwangpo(2000) reported the perithecium of as either oval-shaped or egg-shaped consisting of numerous numbers ofthin long asci containing ascospores Shin (2004) alsoobserved that the apex of the stroma contains numerousperithecia with several ascospores inside asci The ascosporeswere characterized by a thread-like structure in the middlewith non disarticulating part spores attached on both sidesSung (2007) observed perithecia of assuperficial to completely immersed ordinal or oblique inarrangement Asci were hyaline cylindrical and ascosporeswere cylindrical multiseptate disarticulating into part-spores or non-disarticulating Observations of Xie(2010) and Zhong (2010) were also similar to the above

study where they found thread like ascospores in ascus Sung(2010) while working with

observed that the perithecia were ovoid in shape and semi-immersed on the stroma and the ascospores were unevenlyseptate but do not disarticulate into part-spores

The fungus was isolated on PDA medium from the fruitingbodies collected during the survey This showed that thePotato Dextrose Agar which is the most widely used mediumfor fungal isolation can be used for isolation of

Isolation in PDA medium was alreadyreported by many scientists like Sung

Molecular identification of the isolate was done bycomparison of ITS sequences at species level Sequenceanalysis of the culture showed homology with

strain KC1 having 98identity strain KC1 was reported from TamilNadu by Chinnusamy and Krishnamoorthi in 2017 (NCBIGenBank httpncbinlmnhmgovblast) Hence the presentstudy was the first report of from Kerala

The fungus was isolated from stroma stipe and sclerotia offungal-coconut root grub complex on PDA media followedby periodic sub culturing and maintenance of the isolate inPDA slants under refrigerated condition for further studiesDetailed morphological characterization of the isolate wasdone The isolate was identified at species level as

Further studies have to becarried out to unravel the medicinal properties of this fungusand there should be investigations on anti-fungal anti-bacterial and biocontrol properties along with detailed studieson mass production of this precious fungus

Authors are grateful to Kerala Agricultural UniversityThrissur Kerala India for providing facilities to carry out thisstudy in the Department of Plant Pathology at College ofHorticulture Vellanikkara and College of AgriculturePadannakkad

Arora R K 2015 (Berk) Sacc- anentomophagous medicinal fungus-a review

0161-0170

Arora R K Singh N and Singh R P 2013Characterization of an entomophagous medicinalfungus (Berk) Sacc ofUttarakhand India 195-200

Chinnusamy S and Krishnamoorthy A S 2017Identification of 3deoxyadenosine (Cordycepin)

O sinensiset al

et alO sinensis

Cordyceps

et alCordyceps sinensis

et alCordyceps militaris

viz

et al

Ophiocordycepset al

Cordyceps militaris

Ophiocordyceps

C sinensis

et al

et al Ophiocordyceps

et alet al

et al Cordyceps cardinalis

Ophiocordyceps

Ophiocordyceps neovolkianaO neovolkiana

O neovolkiana

Ophiocordyceps neovolkiana

Cordyceps sinensisInt J

Adv Multidiscip Res

Cordyceps sinensis The bioscan

the hyphae w thin-walled branchedaround148 - 161μmbroad

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

2

8

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Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

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(2) 69-76

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(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

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(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

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60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora

Page 6: Observations on entomopathogenic fungus on Coconut root ... Observations o… · observations.These fruit bodies were preserved fresh as well asindryforms. Isolation of fungus was

from the medicinal mushroomsspp (3) 788-792

Dattaraj H R Jagadish B R Sridhar K R and Ghate SD 2018 Are the scrub jungles of Southwest Indiapotential habitats of 20-22

Gwangpo K 2000 Characteristics of species(5) 537-539

Holliday J and Cleaver M 2004 On the Trail of the YakAncient in the modern world

Ko Y F Liau J C Lee C S Chiu C Y Martel J Lin CS Tseng S F Ojcius D M Lai H C and YoungJ D 2017 Isolation culture and characterization of

mycelium from caterpillarfungus fruiting body (1)1-21

Kobayasi Y and Shimizu D 1980 species fromJapan 2

77-96

Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub

Burm(3A) 614-618

Li Y Wang X L Jiao L Jiang Y Li H Jiang S PLhosumtseiring N Fu S Z Dong C H Zhan Yand Yao YJ 2011 A survey of the geographicdistribution of

(6) 913-919

Nam Sung-Hee Li Chun-Ru Hong In-Pyo Sung Kyu-Byoung Kang Seok-Woo Fan Mei-Zhen Li andZeng-Zhi 2006 Isolation and Culture ofE n t om opa t hog en i c Fun gus

(2) 57-61

Pathania P Joshi M and Sagar A 2015 MorphologicalPhysiological and molecular studies on wildlycollected from North WestHlmalayas India (1)53-62

Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146

Sharma S 2004 Trade of from highaltitudes of the Indian Himalaya Conservation andbiotechnological priorities 1614-1619

Shin J C Shrestha B Lee W H Park Y J Kim S YJeong G R Kim H K Kim T W and Sung J M2004 Distribution and favorable conditions formycelial growth of in Korea

(2) 79-88

Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in

(2) 69-76

Shrestha B Weimin Z Yongjie Z and Xingzhong L2010 What is the Chinese caterpillar fungus

( ) (4) 228-236

Sridhar K R and Karun N C 2017 Observations onin the Western Ghats

(2) 104-111

Sung G H Hywel-Jones N L Sung J M Luangsa-ArdJ J Shrestha B and Spatafora J W 2007Phylogenetic classification of and theclavicipitaceous fungi 559

Sung G H Shrestha B Han S K and Sung J M 2011Cultural Characteristics of

Collected from Korea (1)1-6

Sung G Shrestha B Han S K Kim S Y and Sung J M2010 Growth and Cultural Characteristics of

Collected from Korea (4) 274-281

Xie F Zhu Z X Wei K L Zhang S X Zhang N andTian W 2010 Effects of different culture media onthe hyphal growth of strains

(6) 534-536

Zhong X Peng Q Y Qi L L Lei W and Liu X 2010rDNAtargeted PCR primers and FISH probe in thedetection of hyphae andconidia (2) 188-193

OphiocordycepsInt j chem stud

Cordyceps KavakaCordyceps Int

J Med Mushrooms

Cordyceps Onlineposting

Hirsutella sinensisPlos One

CordycepsBulletin of the National Science Museum

Series B BotanyCordyceps

Leucopholis coneophora J Environ ResDev

Ophiocordyceps sinensis JMicrobiol

Cordy c e pssphecocephala Int J Ind Entomol

Cordyceps militarisEur J Biotechnol Biosci

Afr JMicrobiol Res

Cordyceps sinensis

Curr Sci

Cordyceps pruinosaKorean J Mycol

Cordyceps militaris Mycobiol

Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol

Ophiocordyceps nutans JNew Biol Reports

CordycepsStud Mycol

Ophiocordycepsheteropoda Mycobiol

Cordyceps cardinalisMycobiol

Cordyceps sinensisChinese J Microecol

Ophiocordyceps sinensisJ Microbiol Methods

5

51

11

12

8

49

13

3

7

86

32

33

1

6

57

39

38

22

83

60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora