R ESEARCH ARTICLEdoi: 10.2306/scienceasia1513-1874.2020.078

ScienceAsia 46 (2020): 557–567

Morphological study, production and cryopreservationof blastospores of entomopathogenic fungiLakkhana K. Wingfield∗

Division of Biological Science, Faculty of Science, Prince of Songkla University, Songkhla 90110 Thailand

e-mail: Lakkhana.k@psu.ac.thReceived 11 Jul 2020Accepted 7 Oct 2020

ABSTRACT: Entomopathogenic fungi have been known as potential candidates for insecticides and metaboliteproducers. The aims of this study were to investigate characteristic and development patterns of blastosporesand to determine optimal culture conditions for blastospore production of entomopathogenic fungi in the generaAkanthomyces, Cordyceps, Hirsutella, Metarhizium, and Torrubiella. Production of blastospores was induced usingGrace’s insect cell medium (GICM) supplemented with foetal bovine serum (FBS) except the genus Torrubiella. Out of39 fungal isolates, 18 isolates could produce blastospores, most of them belonged to the genus Cordyceps. Observationon characteristics and formation of blastospores revealed that blastospores produced from all genera shared commoncharacters and formation. However, some distinctive characters were observed in the genus Akanthomyces. Effects ofculture media, types of inoculum and cultivating condition were examined for the production of highest numbers ofblastospores (up to 107–108 blastospores/ml), revealing different requirements in each fungal isolate for the productionof blastospores. Cryopreservation of blastospores revealed that freeze-drying method could be used to preservedblastospores of C. brongniartii, Hisutella sp. 02 and Metarhizium sp. 02 except A. pistillariiformis. This study is thefirst comprehensive investigation on development patterns of blastospores and factors underpinning effective cultureconditions for blastospores production of the selected entomopathogenic fungi.

KEYWORDS: entomopathogenic fungi, blastospores, cryopreservation

INTRODUCTION

Entomopathogenic fungi are important natural reg-ulators of insect populations as they can generallyinfect a broad range of insects and cause mortal-ity of the insect hosts. Since they are considerednatural mortality agents and environmental safe,the fungi have been proposed as a group of poten-tial candidates for microbial insecticides to controlagricultural pests [1–4]. For example, Beauveriabassiana 36 was investigated for its virulence andpathogenicity on the infected larvae of Helicoverpaarmigera, the most serious pest of several plantspecies, in the recent report [5]. Entomopathogenicfungi have also been signified as a valuable source ofsecondary bioactive metabolites. Several bioactivecompounds have been isolated from Cordyceps spp.,for instance, antimalarial cordypyridones A-D fromC. nipponica [6, 7], bioxanthracenes from C. pseu-domilitaris [7], antimalarial red naphthoquinonesfrom C. unilateralis [8], and beauvericin-like com-pound from C. militaris [9].

The entomopathogenic fungi consist of varioustaxa and do not form a monophyletic group. Most

of common and important entomopathogenic fungiare grouped in the order Hypocreales which be-longs to the phylum Ascomycota. These includethe anamorphic phases (Beauveria spp., Hirsutellaspp., Metarhizium spp., Nomuraea spp., and Pae-cilomyces spp.) and the teleomorphic phase (Cordy-ceps spp.). Meanwhile, other entomopathogenicfungi (Entomophthora spp., Entomophaga spp., Pan-dora spp., and Zoophthora spp.) belong to theorder Entomophthorales of the phylum Zygomy-cota. Mode of production of asexual and sexualpropagules has been proposed as a key basis foridentification of these fungi. Besides the vegetativepropagules, other structures, such as rhizoids, rest-ing spores, hyphal bodies (including protoplasts andblastospores), and sclerotia are also important forthe classification of a fungal species or a genus [10].Unlike other fungal groups, the entomopathogenicfungi can produce secondary spore-type which iscommonly referred to blastospore [11, 12].

Formulation of microbial insecticides fromentomopathogenic fungi often centers on mass-production of aerial conidia [1–3]. Interestingly, ithas been evidenced that blastospores demonstrated

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stronger infection ability than conidia in terms offaster germination rate on the insect cuticle andhigher level of bioactivity and insecticidal efficacyagainst various insect pests [13–15], which thusmake blastospores the propagules of choice for theproduction of a biological insecticide. However, invitro cultivation of blastospores is still very fastidi-ous in some fungal genera because traditional cul-ture conditions for fungal growth are unfavourablefor the production of blastospores thus different pa-rameters are required [16]. In addition, informationon blastospore development and characteristics ofindividual entomopathogenic fungal genus is stillsparse, which hinder the use of blastospores asmicrobial pesticides.

Since entomopathogenic fungi are a rich sourceof natural bioactive compounds and demonstratecertain advantages over the use of chemical in-secticides, production of the microbial insecticides,particularly exploitation of the blastospore as anactive ingredient, is challenging. In addition, un-derstanding the production and development pat-terns of blastospores of the fungi is also crucial fordeveloping blastospores as microbial insecticides.In response to those challenges, culture conditionsfor the production of blastospores, including blas-tospore development patterns, should be defined.The aim of the present study was to investigatecharacteristics and development patterns of blas-tospores of the selected entomopathogenic fungalgenera. In addition, culture conditions to induce theproduction of blastospores were also determined.Besides difficulties in producing blastospores formorphological study and mass-production for indus-trial use, storage of fungal cultures is also very im-portant that these cultures should be preserved in aphysiologically and genetically stable state in orderto maintain their valuable properties. Therefore,long-term preservation method via freeze-drying ofthe blastospores were also investigated.

MATERIALS AND METHODS

Fungal isolates and maintenance

Fungal isolates of the entomopathogenic fungi usedin the present study were from the genera Akan-thomyces, Cordyceps, Hirsutella, Metarhizium, andTorrubiella. All fungal isolates were maintainedon Potato Dextrose Agar (PDA) (HiMedia®) andincubated at 25 °C for 10–15 days.

Induction and observation of blastosporedevelopment patterns and morphology

Optimum concentrations of the foetal bovine serum(FBS; GIBCO®, USA) used for the productionof blastospores were determined. Ten ento-mopathogenic fungal isolates belonged to five fun-gal genera (Akanthomyces pistillariiformis, Akan-thomyces sp. 01, Cordyceps brongniartii, Cordycepssp. 01, Cordyceps sp. 02, Hirsutella sp. 01, Hir-sutella sp. 02, Metarhizium sp. 01, Metarhiziumsp. 02, Torrubiella sp. 01, and Torrubiella sp. 02)were selected for the study. A mycelial plug(0.5 mm in diameter) of each entomopathogenicfungal strain was transferred into 1 ml of Grace’sinsect cell medium (GICM; GIBCO®, USA) con-taining 0.60 g/l L-glutamine, 3.33 g/l lactalbuminhydrolysate, 3.33 g/l yeastolate, and 3% (w/v)glucose and supplemented with different concentra-tions of FBS (0, 1, 5 and 10% (v/v)) in a 24-wellmicrotitre plate. The cultures were incubated at28 °C under static condition and the formation ofblastospores was investigated daily for 14 days usinginverted microscope. Formation, characteristics andmorphological development of blastospores wereobserved daily using an inverted microscope.

Large scale cultivation of blastospores

To monitor rate of blastospore production, ei-ther conidia suspensions or mycelial plugs wereused. Conidia suspensions (103 conidia/ml) wereprepared using sterile distilled water containing1% (v/v) of tween 80. Conidia suspensions (200 µl)or 5 mycelial plugs, were transferred into 5 ml ofyeast extract-peptone-glucose (YPG) broth or GICMin a 6-well plate and incubated at 28 °C understatic condition and shaking condition at 100 rpm.The numbers of blastospores were counted usinghaemacytometer (Boeco, Germany). The experi-ment was conducted in triplicate. The conditionsgiving blastospore concentration of more than 108

blastospores/ml were selected for the productionof blastospores to be used in the cryopreservationprocess.

Cryopreservation of blastospores ofentomopathogenic fungi

Blastospores were filtered through double layers ofsterile Miracloth and adjusted to desired concentra-tion of 107–108 blastospores/ml. Each blastosporesuspension was centrifuged at 6 600 rpm for 5 minto remove the culture media and the pellet washedtwice with sterile distilled water. The resulting blas-

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Table 1 Determination of FBS concentration for blas-tospore production (blastospores/ml).

Fungal isolateFBS concentration (%)

0 1 5 10

A. pistillariiformis 102 105 108 >108

Akanthomyces sp. 01 – – – –C. brongniartii 105 107 108 >108

Cordyceps sp. 01 104 106 107 107

Hirsutella sp. 01 – – 10 102

Hirsutella sp. 02 – – 102 104

Metarhizium sp. 01 – – 102 104

Metarhizium sp. 02 10 104 106

Torrubiella sp. 01 – – – –Torrubiella sp. 02 – – – –

tospore pellets were used within 2 h after washing.Protective agent (5% (w/v) trehalose) was preparedin 0.1 M potassium phosphate buffer (pH 7.0) andsterilized at 115 °C for 15 min. Blastospore pelletwas resuspended in 0.1 ml of the protective agentand filled in an ampoule. Primary vacuum dryingwas performed for the minimum of 3 h, followed bysecondary drying at 0.090 mbar at −60 °C for 20 h.Ampoules were sealed and stored at 5 °C in the dark.

Blastospore viability was evaluated before dry-ing, immediately after drying and after one-yearstorage. Freeze-dried blastospores were dispersedin 0.1 ml rehydration fluid (peptone 5 g, yeastextract 3 g, MgSO4 1 g, and distilled water to 1 l,pH 7.0). Rehydrated blastospores were transferredinto microtitre plate wells containing 1.5 ml ofGICM. After 8–15 h of incubation, one drop of 2 NHCl was added to halt the germination process. Ger-mination of blastospores was assessed microscopi-cally; and counting was done in duplicate from twoampoules. Revived cultures were also observed formorphological and genetic stability, such as changesin pigmentation or colony morphology.

RESULTS

FBS concentration for the production ofblastospores

Ten entomopathogenic fungal isolates belonged tofive fungal genera (Akanthomyces, Cordyceps, Hir-sutella, Metarhizium, and Torrubiella) were selectedfor the study. Mycelial plugs of each isolate weregrown in GICM supplemented with 0, 1, 5 and10% (v/v) FBS. The results revealed that blas-tospore production was observed from the genusCordyceps and A. pistillariiformis without addition ofFBS (Table 1). With addition of FBS, the production

Table 2 Screening of blastospore production from 39entomopathogenic fungal isolates.

GeneraNo. of isolates tested/No. of blastosporeproducing isolates

Frequency ofblastosporeproduction (%)

Akanthomyces 9/3 33Cordyceps 16/10 63Hirsutella 5/3 60Metarhizium 5/2 40Torrubiella 4/0 0

of blastospores was significantly enhanced. FBSsupplementation was necessary in the genera Hir-sutella and Metarhizium as blastospore productionwas induced at high concentration of FBS (5 and10% (v/v)). Meanwhile, none of the Torrubiellaand Akanthomyces sp. 01 isolates produced blas-tospores. This might infer specific preference ofeach fungal strain for blastospore production invitro. Although 5% (v/v) FBS was sufficient for mostisolates, 10% (v/v) FBS was chosen for the entirestudy based on satisfactory blastospore yield.

Screening for fungal isolates capable forblastospore production

Production of blastospores was screened from 39entomopathogenic fungi using GICM supplementedwith 10% (v/v) FBS. Out of 39 fungal isolates, 18isolates were able to produce blastospores (Table 2).Most of them belonged to the genera Cordycepsand Hirsutella (10/16 and 3/5, respectively) whileblastospore production was detected in few isolatesof Akanthomyces and Metarhizium. In addition,none of Torrubiella isolates produced blastosporesin GICM supplemented with 10% (v/v) FBS.

General characteristics and formation patternsof blastospores

Observation on characteristics and formation pat-terns of blastospores revealed that blastospores pro-duced from all genera shared common charactersand formation patterns (Table S1). However, somedistinctive characters could be observed in somefungal isolates. In the Akanthomyces spp., blas-tospore formation was generally observed after 1–2 days of incubation. The spores were hyaline,small, round or oval shapes with 4-10 µm averagein length. They were usually produced from hyphaltips and formed clusters. After released into themedium, blastospores immediately produced a newbudding cell. In addition, it can be observed that

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Akanthomyces spp. demonstrated unique character-istics of blastospores that differed from other generain terms of shape, size and budding position on thehypha. In the Cordyceps spp., blastospore formationwas generally observed after 2–3 days of incubation.The spores were hyaline, ellipsoidal to cylindricalshapes which connected to the mycelium by a shortneck. Average length of blastospore was 10–25 µm.In addition, the spores usually produced from hy-phal tips and sometimes from sides. In the Hirsutellaspp., blastospore formation was generally observedafter 2–5 days after inoculation depending on fungalstrains. The spores were mainly hyaline, small,narrow with cylindrical to club-shapes. However,some blastospores were ellipsoidal, cylindrical tolong rod shapes. The length of blastospores wasbetween 15–20 µm. Blastospores of Hirsutella werefound to be generated very rapidly by budding bothfrom the mother cells and from the hyphal sides.In the Metarhizium spp., blastospore formation wasgenerally observed after 2–3 days of incubation.The spores were hyaline, ellipsoidal to cylindricalshapes and usually produced from hyphal sides withan attachment through a short neck. Average lengthof blastospore was 10–20 µm.

Detailed morphological development patternsof blastospores

Representative isolates of each fungal genus(C. brongniartii, Hisutella sp. 02, Metarhiziumsp. 02, and A. pistillariiformis) were chosenas model organisms, based on their ability toproduce large numbers of blastospores and theirindustrial importance, to study the morphologicaldevelopment of blastospores.

C. brongniartii is one of the most importantspecies among Cordyceps spp. This fungus can pro-duce cuticle-degrading peptidases, which can po-tentially be used in agricultural and medical ap-plications. Colony appearance of C. brongniartiigrown on PDA at 28 °C for one week were woollyto powdery with approximately 19.3±1.6 mm indiameter (Fig. 1a). Colony color was white tocream and yellowish-brown to dark brown on thereverse side resemble to its anamorph. After threeweeks of incubation, the colony changed to brownand slender stalks of stromata were produced onthe colony (Fig. 1b). Conidia were abundant, el-liptical, with thick smooth walls and borne alongthe sides and ends of repeatedly branched hy-phae to form large clusters. Size of conidia was10.3±1.4 µm×4.7±0.5 µm (Fig. 1c). Blastosporeformation of C. brongniartii was observed daily

in GICM supplemented with 10% (v/v) FBS andthe developmental process was initially examinedfrom the stage of conidia development. The on-set of conidia germination was marked by grad-ual swelling of conidia until it was approximately30–100% larger than the original size after 8 h ofincubation (Fig. 1d). After that the emergence ofunipolar germ tubes were detected at 12 h. Fur-ther elongation of the germ tubes and septationof hypha were apparent after 16 h of incubation(Fig. 1e). During this stage, bipolar or multipolargrowth of young mycelia from the conidium oc-curred. After elongation of mycelia, a blastosporeappeared laterally or sometimes terminally frommycelia after 24 h of incubation. Blastospores wereconnected to mycelium by a short neck. Thesespores propagated by production through a poreand bulged out near the septa. Release of free blas-tospores occurred after 36 h of incubation (Fig. 1f).At this stage, blastospores were hyaline, single-celled with thin smooth-wall resemble protoplast-like cells. Detached blastospores were mostly ellip-tical shape. The average sizes of blastospores were10.9–23.4 µm×3.1–3.2 µm. Then, the spores’ elon-gation proceeded and a cross-walled was made toseparate part of the stretched blastospores after 48 hof incubation (Fig. 1g). These short mycelia derivedfrom blastospores could reproduce new blastosporeson the third day of cultivation (Fig. 1(h,i,j)). Newgeneration of blastospores could be obtained fromreproduction of newly grown mycelia.

Colony appearance of Hirsutella sp. 02 grownon PDA at 28 °C for 7 days was flat with shortaerial mycelia and moist colony surface with ap-proximately 14.24±1.13 mm in diameter (Fig. 2a).Colony color was cream to golden brown and darkbrown on the reverse side. Blastospores were hya-line, single-celled with smooth-wall and appearedlaterally or sometimes terminally from mycelia after2 days of incubation (Fig. 2b). The release of freeblastospores was observed after 2.5 days of incuba-tion. Detached blastospores were mostly ellipticalto rod shape. The average size of detached blas-tospores was 11.1–22.6 µm×3.6–4.0 µm (Fig. 2c).After release of blastospores, the spores were elon-gated and a cross wall was made to separate partof the stretched blastospores after 3.5 days of in-cubation (Fig. 2d). However, these short myceliaderived from blastospores could not reproduce newblastospores as observed in C. brongniartii. In thiscase, new blastospores were generated from bud-ding of the mature blastospores only.

Metarhizium spp. is one of the most potential

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Fig. 1 Characteristics and morphological development patterns of blastospores of C. brongniartii. The liquid culture wasgrown in GICM supplemented with 10% FBS and incubated at 28 °C under static condition. (a) Colony of C. brongniartiion PDA incubated at 28 °C for 7 days; (b) colony after incubation for 3 weeks; (c) conidia; (d) gradual swelling ofconidia and emergence of a unipolar germ tube at 8–12 h; (e) blastospore formation from germinating conidia at 16–24 h; (f) released blastospores at 36 h; (g) germination and elongation of blastospores at 48 h; (h,i,j) generation ofblastospores from a mature mycelium at 54 h.

Fig. 2 Characteristics and morphological development patterns of blastospores of Hirsutella sp. 02. The liquid culturewas grown in GICM supplemented with 10% FBS and incubated at 28 °C under static condition. (a) Colony of Hirsutellasp. 02 on PDA incubated at 28 °C for 7 days; (b) blastospore formation after 2 days of incubation; (c) releasedblastospores after 2.5 days of incubation; (d) germination and elongation of blastospores after 3.5 days of incubation.

entomopathogenic fungi which is used as biologi-cal insecticide to control a number of pests, suchas grasshoppers, termites, thrips, etc. Colony ap-pearance of Metarhizium sp. 02 grown on PDA at28 °C for one week was flat and velvety with ap-proximately 22.61±2.15 mm in diameter (Fig. 3a).Colony color was white to creamy and yellow to or-ange on the reverse side. Conidia were single-celled,

cylindrical or ovoid, forming chains (Fig. 3b). Blas-tospores were observed after 2 days of cultiva-tion and appeared laterally or sometimes terminallyfrom mycelia. They were hyaline, single-celledwith smooth-wall (Fig. 3cd). Detached blastosporeswere mostly elliptical to rod shape with the averagesize of 15.0–21.3 µm×3.6–4.7 µm. After releasedinto the medium, enlargement of blastospores oc-

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Fig. 3 Characteristics and morphological development patterns of blastospores of Metarhizium sp. 02. The liquidculture was grown in GICM supplemented with 10% FBS and incubated at 28 °C under static condition. (a) Colonyof Metarhizium sp. 02 on PDA incubated at 28 °C for 7 days; (b) conidia; (c,d) blastospore formation after 2 days ofincubation; (e,f) germination and elongation of blastospores after 3 days of incubation.

Fig. 4 Characteristics and morphological development patterns of blastospores of A. pistillariiformis. The liquidculture was grown in GICM supplemented with 10% FBS and incubated at 28 °C under static condition. (a) Colonyof A. pistillariiformis on PDA incubated at 28 °C for 7 days; (b) blastospore formation after 2 days of incubation;(c,d) released blastospores.

curred, followed by a production of germ tubes after3 days of incubation. A cross wall was subsequentlymade to separate part of the stretched blastospores.During this stage, shape of blastospores were trans-formed to tamarind-like shapes and size was in-creased simultaneously (Fig. 3e). After 5 days ofcultivation, germinated blastospores became shortmycelia and continued to grow slowly in the GICM.However, formation of new blastospores was notdetected in this study (Fig. 3f).

Members in the genus Akanthomyces can pro-duce valuable toxins and metabolites. Colony ap-pearance of A. pistillariiformis grown on PDA at28 °C for one week was flat and wet on the surfacewith approximately 12.78±2.05 mm in diameter(Fig. 4a). Colony color was cream to white and yel-low on the reverse side. Mycelia grew very slowly inthe liquid medium. Blastospores were observed af-ter 2 days of cultivation, which appeared laterally or

sometimes terminally from mycelia (Fig. 4b). Theywere hyaline, single-celled with smooth-wall withvarying shapes from round to oval (Fig. 4c). Theaverage size of blastospores was 4.3–8.1 µm×3.9–4.3 µm and usually produced from hyphal tips andformed clusters. After released into the medium,blastospores began to bud immediately (Fig. 4c).However, new budding cells did not detach from themother cells which were not the case in C. brong-niartii and Hirsutella sp. 02. No germination ofblastospores was observed within 14-day period ofobservation (Fig. 4d).

According to the observation of developmentalprocess of blastospores, common characteristics ofblastospores among the isolates can be summarized.Generally, blastospores are single cells, hyaline,and non-motile with smooth thin-walled resemblingprotoplast-like cells. They propagated by burstingthrough a pore on a hyphal fragment and connected

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to the mycelium through a short neck. Once rami-fication of mycelium has taken place under suitableenvironmental conditions, formation of blastosporewas induced. After disconnected, blastospores en-larged in size and shape, followed by initiationof germination to form new mycelia. However,blastospores of some genera underwent through aprocess of budding similar to that of yeasts.

Effects of media, types of inoculum andcultivating conditions on the production ofblastospores

Optimal condition for the production of blastosporesof C. brongniartii, Hirsutella sp. 02, Metarhiziumsp. 02, and A. pistillariiformis were determined.Effects of media, types of inoculum and cultivat-ing conditions were examined for the maximumproduction of blastospores. Two different media(GICM and YPG) were tested. The results showedthat blastospore production of C. brongniartii wasinduced by cultivation in both GICM and YPG liquidmedia (Fig. 5a). The GICM gave a higher yield ofblastospores compared to that of YPG regardlessof types of inoculum and cultivating conditions.Meanwhile blastospore production of Hirsutella sp.02, Metarhizium sp. 02 and A. pistillariiformis couldonly be induced by cultivation in GICM. Therefore,the GICM was used as culture medium in all subse-quent experiments in this study.

Two different types of inoculum (mycelial plugand conidia suspension) were also tested. The re-sults showed that conidia suspension gave a slightlyhigher yield of blastospores than that of mycelialplug when cultivated in the GICM for all testedfungal isolates (Fig. 5a–d). Numbers of blastosporesproduced from both types of inoculum were not sig-nificantly different according to Duncan’s multiplerange test (p < 0.05). Therefore, mycelial plugswere used as a starting inoculum for the productionof blastospores due to cost and ease of preparation.Different cultivating conditions, shaking and staticwere subsequently evaluated. Regardless of inocu-lum types and culture media, production rate ofC. brongniartii blastospores under static conditionincreased slowly; and the numbers of blastosporesobtained were lower when compared to those undershaking condition. However, cultivating conditionswere species-specific as no blastospore was observedunder shaking condition from Hirsutella sp. 02,Metarhizium sp. 02 and A. pistillariiformis cultures.

Time course for the production of blastosporeswas observed in all entomopathogenic fungal iso-lates. In the C. brongniartii, blastospore formation

initiated at 12–18 hours post inoculation (hpi) andyield of blastospores reached the maximum level at36–72 hpi. After 78 hpi of cultivation, most of blas-tospores started to germinate. Regarding cultivatingconditions, the highest yield of blastospores was2.43 × 106 blastospores/ml under static conditionwhen either conidia or mycelial plugs were used asinoculum in GICM. On the other hand, the highestyield of blastospores under shaking condition was3.28–4.35×108 blastospores/ml when either coni-dia or mycelial plugs were used as inoculum inGICM. It can be thus concluded that the best condi-tions for the production of blastospore of C. brong-niartii was using GICM as the induction mediumunder shaking condition. The suitable time coursefor harvesting fresh blastospores, to be used asspecimens for cryopreservation, was approximatelyat 42–72 hpi after incubation.

In Hirsutella sp. 02, blastospore formation wasinitiated at 36 hpi and numbers of blastospores wereexponentially increased within a day. A suddendecrease in spore production rate was subsequentlydetected, followed by second period of exponentialincrease in numbers of blastospores. This inter-esting observation indicated a unique pattern ofblastospore development of Hirsutella sp. 02 in theGICM. The sudden change in the numbers of blas-tospores at 84 hpi could arise from the fact thatmajority of budding blastospores were detachedfrom one another, resulting in an increased numbersof blastospores. Blastospore production reached themaximum level at 96–132 hpi, yielding 4.8×106blastospores/ml. A decline in blastospore numbersoccurred immediately due to rapid germination ofmost blastospores. According to the results, the bestconditions for the production of blastospore of Hir-sutella sp. 02 were cultivating in GICM under staticcondition. The suitable time course for harvestingfresh blastospores was approximately at 84–144 hpi.

In Metarhizium sp. 02, blastospore formationwas initiated at 48 hpi. The exponential increasein blastospore numbers was observed within 72 hpias budding blastospores were detached from oneanother. The highest numbers of blastosporeswas observed at 108 hpi, yielding 4.4×106 blas-tospores/ml. A sharp decline in blastospore num-bers was observed after 4 days of incubation re-sulting from rapid formation of emerging youngmycelia. It can be thus concluded that the bestconditions for the production of blastospores ofMetarhizium sp. 02 were using GICM as the in-duction medium under static condition. The suit-able time course for harvesting fresh blastospores

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Fig. 5 Production of blastospores of selected entomopathogenic fungi incubated at 28 °C under shaking and staticconditions. Mycelial plugs and conidia were used as stating inocula. (a) Production of blastospores of C. brongniartii inYPG (dotted line) and GICM (solid line); (b) production of blastospores of Hirsutella sp. 02 in GICM; (c) production ofblastospores of Metarhizium sp. 02 in GICM; (d) production of blastospores of A. pistillariiformis in GICM. The resultsare presented as the mean±SD of three independent experiments.

was approximately at 84–144 hpi. In A. pistil-lariiformis, blastospore formation was initiated at12 hpi and numbers of blastospores were expo-nentially increased up to 48 hpi, yielding 8.8×107blastospores/ml. Therefore, the best conditions forblastospore production of A. pistillariiformis werecultivating in GICM under static condition. The suit-able time course for harvesting fresh blastosporeswas approximately at 24–72 hpi after incubation.

Cryopreservation of blastospores ofentomopathogenic fungi

Long-term preservation of blastospores of the en-tomopathogenic fungi was studied. Freeze-dryingwas used as a preservation method and 5% (w/v)trehalose was used as a protective agent. Viability

Table 3 Viability of four entomopathogenic fungi usingfreeze-drying method.

StorageViability (%)

Preservation One-year storage

C. brongniartii 31.8±3.4 21.8±1.8aHirsutella sp. 02 41.8±2.2 35.6±0.9bMetarhizium sp. 02 31.8±2.2 22.0±1.7cA. pistillariiformis 0.00±0.0 0.00±0.0

The results are presented as the mean value±SDof three independent experiments. Different lettersindicate significant differences according to Duncan’smultiple range test (p < 0.05).

of blastospores was assessed by determination ofgerm tube formation assay immediately after preser-

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vation and after one-year storage at 5 °C in thedark. The preservation results revealed that freeze-drying method could be used to preserve fungalblastospores of C. brongniartii, Hisutella sp. 02 andMetarhizium sp. 02 because satisfactory percentageof viability was obtained after one-year storage, asshown in Table 3. However, the method was notsuitable for preserving blastospores of A. pistillari-iformis since no viability was observed. Observa-tion of revived cells in terms of morphological andgenetic stability demonstrated no changes in pig-mentation, colony morphology including ability toproduce blastospores and reproductive structures.

DISCUSSION

Entomopathogenic fungi in the genera Akan-thomyces, Cordyceps, Hirsutella, Metarhizium, andTorrubiella have been recognized as potential can-didates for the production of microbial insecticidesto control several agricultural pests and have beenidentified as valuable sources of bioactive metabo-lite production. They are considered agricultur-ally and medically important. Therefore, under-standing the morphological development of ento-mopathogenic fungi is one of the key parametersprior to using blastospores in industrial production.In addition, preservation methods which maintaingenetic and physical stabilities of entomopathogenicfungi, were required to avoid alterations duringstorage.

In the present study, production of ento-mopathogenic fungal blastospores could be inducedby using GICM as a culture medium supplementedwith FBS. However, induction of blastospores invitro depends on fungal genera since the findingsrevealed that none of the isolates in the genusTorrubiella could produce blastospores. Similarly,production of blastospores also depends on fungalisolates since blastospore formation could be de-tected in some isolates of the genera Akanthomyces,Cordyceps, Hirsutella and Metarhizium with differ-ent efficiency. As blastospore formation has beenreported as an indispensable phase in the fungalreproducing cycle [17], failure in the productionof blastospores in some fungal isolates could beattributed to inappropriate conditions of cultivationused.

Out of 39 entomopathogenic fungal isolates, 18isolates were able to produce blastospores. Mostof them belonged to the genera Cordyceps and Hir-sutella. Most isolates of Cordyceps spp. were capa-ble to produce blastospores (63%) which suggestedthat Cordyceps spp. is non-fastidious and does not

require complex nutrients for growth and devel-opment. Common characteristics of blastosporesamong those four genera were single-cell, hyaline,non-motile with smooth thin-walled, and variedin size and shape. Development patterns of blas-tospore formation also depends on fungal generain term of day of spore initiation, spore emergingposition, spore budding, and spore germination.It can be noted that blastospore morphology anddevelopment of Akanthomyces sp. is unique amongother genera. This characteristic could be usedto distinguish Akanthomyces sp. from other fungalgenera. This corresponds to a statement from Sam-son et al [9] who stated that blastospores may beused to classify a genus of entomopathogenic fungiin the same manner as other vegetative propagulesor other structures.

Culture media, types of inoculum and culti-vating conditions exhibited different impacts onthe production of blastospores. The GICM supple-mented with FBS could be used to induce the pro-duction of blastospores in all fungal genera tested,however, YPG medium could only be used to in-duce the production of blastospores in the genusCordyceps (C. brongniartii). FBS acts to mimicnatural condition of haemolymph and contains es-sential components, such as hormones, vitamins,minerals, trace elements, and growth factors [18],which contributed to the induction of blastosporeformation of entomopathogenic fungi in vitro. Anability of C. brongniartii for production blastosporesin YPG medium might be attributed from non-fastidious character of the fungus including strongvirulence resembling to its anamorph, B. brong-niartii. This ability could thus be one of the fac-tors contributing to adaptation ability to diverseenvironmental conditions. YPG medium was alsoused to promote the production of blastosporesin B. bassiana and C. brongniartii under shakingcondition, yielding 8.5×107 blastospores/ml and5.7×106 blastospores/ml, respectively, after 2 daysof incubation [19].

In addition to culture media, either mycelialplugs or conidia suspensions could be used as start-ing inocula since the numbers of blastospores pro-duced from both were not significantly different.Cultivating conditions also showed different im-pacts on blastospore production because cultivationunder static condition was only suitable for Hir-sutella sp. 02, Metarhizium sp. 02 and A. pistillari-iformis, while C. brongniartii produced blastosporesin both static and shaking conditions. Productionof blastospores under shaking condition has been

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566 ScienceAsia 46 (2020)

described as a suitable condition for the produc-tion of blastospores in several entomopathogenicfungi, for example M. flavoviride, B. bassiana, P. fu-mosoroseus, and C. unilateralis) [20–23]. However,in the present study, production of blastospores ofHirsutella sp. 02, Metarhizium sp. 02 and A. pistil-lariiformis was only successful under static condi-tion. Furthermore, those parameters tested also hadsignificant impacts on time course of blastosporeformation and determination of cultivation periodthat generated the highest numbers of blastospores.The observation revealed that initiation of blas-tospore formation of C. brongniartii occurred fasterthan other isolates. This might be a result from thefact that this fungus is fast-growing when grownon PDA and in GICM while the others are slow-growing.

The findings provided not only characteristicsand development patterns of blastospores of en-tomopathogenic fungi, but also demonstrated therequirements for specific nutrients and conditionsfor growth and development of individual isolate.Some fungal species might need different types ofsugars or nitrogen sources in medium composition,which could affect the numbers of blastospore pro-duction, growth and morphology. Formation ofblastospores might be affected by age of the cultureand loss of virulence due to routine subculturing.As blastospores serve as a virulent determinant inentomopathogenic fungi, loss of virulence due toroutine subculturing may result in loss of ability toproduce blastospores accordingly.

Long-term preservation of blastospores usingfreeze-drying method was successful in C. brong-niartii, Hisutella sp. 02 and Metarhizium sp. 02.However, the method was not suitable to preserveblastospores of A. pistillariiformis because no viabil-ity was observed. This finding might suggest dif-ferent compositions and properties of blastosporesof Akanthomyces spp., which make the fungus moresensitive to freeze-drying. This, consequently, leadsto a problem in preservation of this fungus. There-fore, further investigation to identify an appropriatetechnique, conditions and protective agents mustbe conducted for this genus. In addition, severalreports described that selection of optimal growthphase for cryopreservation is also essential for sur-vival of dried spores. For example, the station-ary phase cells of Lactobacillus rhamnosus gave thehighest recovery, after drying, of 31–50%, whereasearly log phase cells exhibited 14% survival and lagphase cells showed the highest susceptibility, withonly 2% cell survival [24]. Similar results were

reported by Mary et al [25], where significantlyhigher cell viability was achieved from stationaryphase Rhizobia cells compared to exponential phasecells. In contrast, higher survival rates of Sinorhi-zobium and Bradyrhizobium were produced whensampled in the lag phase of growth [26]. Accordingto the finding from Cliquet and Jackson [20], adefined medium containing basal salts, glutamate,glucose, and zinc can be used to induce optimalconcentrations of desiccation-tolerant blastosporesof P. fumosoroseus. However, in this study thegrowth phase of entomopathogenic fungi was notdetermined but only the time course for maximumproduction of blastospores.

Appendix A. Supplementary data

Supplementary data associated with this arti-cle can be found at http://dx.doi.org/10.2306/scienceasia1513-1874.2020.078.

Acknowledgements: This work was supported by theNational Science and Technology Development Agency(NSTDA), Thailand and grants from Prince of SongklaUniversity Research and Development Office for LakkhanaKanhayuwa Wingfield.

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Appendix A. Supplementary data

Table S1 General characteristics and blastospore formation patterns of the entomopathogenic fungi.

Genera/species Blastospore morphologySize Days of

formationBuddingposition

Blastospores(µm) (X400 magnification)

A. pistillariiformis hyaline, small, round or ovalshapes

4–10 1–2 hyphal tips(clusters)Akanthomyces sp. 01

Akanthomyces sp. 02

C. brongniartii hyaline, ellipsoidal tocylindrical shapes

10–25 2–3 hyphal sidesC. pseudomilitarisCordydeps sp. 01Cordydeps sp. 02Cordydeps sp. 03Cordydeps sp. 04Cordydeps sp. 05Cordydeps sp. 06Cordydeps sp. 07Cordydeps sp. 08

Hirsutella sp. 01 hyaline, small, narrow withcylindrical to club-shapes

15–20 2–5 hyphal sidesHirsutella sp. 02Hirsutella sp. 03

Metarhizium sp. 01 hyaline, ellipsoidal tocylindrical shapes

10–20 2–3 hyphal sidesMetarhizium sp. 02

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