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
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(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
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Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub
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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
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Sangdee A and Sangdee K 2013 Isolation identificationculture and production of adenosine and cordycepinfrom cicada larva infected with entomopathogenicfungi in Thailand (2) 137-146
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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
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Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in
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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
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Afr JMicrobiol Res
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Curr Sci
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Cordyceps militaris Mycobiol
Ophiocordyceps sinensis OphiocordycipitaceaeMycol Int J Fungal Biol
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32
33
1
6
57
39
38
22
83
60 Observations on entomopathogenic fungus on Coconut root grubOphiocordyceps neovolkiana Leucopholis coneophora
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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59P K Laya C K Yamini Varma K Anita Cherian M M Anees and C R Rashmi
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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
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Kumar S T and Aparna N S 2014 species as abio-control agent against coconut root grub
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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
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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
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Shrestha B Han S K Yoon K S and Sung J M 2005Morphological Characteristics of Conidiogenesis in
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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
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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
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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
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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
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Hirsutella sinensisPlos One
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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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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
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
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
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
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