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Journal of Stored Products Research 47 (2011) 157e160

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Journal of Stored Products Research

journal homepage: www.elsevier .com/locate/ jspr

Germination at different water activities of similarly aged Metarhizium conidiaharvested from ageing cultures

Catherine Smith a, Steve Edgington b,*

aUniversity of Southampton, Highfield, Southampton SO17 1BJ, UKbCABI Europe - UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY, UK

a r t i c l e i n f o

Article history:Accepted 29 January 2011

Keywords:MetarhiziumEntomopathogenic fungusWater activityConidiaGerminationStorage pest

* Corresponding author. Tel.: þ44 (0) 1491 829080E-mail address: s.edgington@cabi.org (S. Edgingto

0022-474X/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jspr.2011.01.007

a b s t r a c t

The ability of two Metarhizium species, M. acridum (IMI 330189) and M. anisopliae (IMI 385045), togerminate at five water activities ranging from 0.998 to 0.944, was examined for conidia harvested fromcultures of different ages. The culture ages ranged from 4 to 22 days old. All of the conidia on each platewere harvested 4 days after inoculation, then again after 11, 18 and 22 days. Therefore, the conidia werenever greater than 7 days old. The data suggested that conidia of M. anisopliae were generally fitter, asdefined by higher viability at all water activities, when harvested from the 4 day-old culture compared tothose from the 22 day-old culture. At the lowest water activity (0.944) germination of M. anisopliaeconidia was 71.5% from the 4 day-old culture compared to 0% from the 22 day-old culture. In contrast,conidia of M. acridumwere generally fitter when they were obtained from 11, 18 and 22 day-old culturesthan the conidia obtained from the 4 day-old culture. However, at the lowest water activity, germinationfor all treatments of M. acridum was less than 8%. The culture growth phase and media age may affectconidia fitness. The capacity of conidia to withstand water stress and possible reasons for interspecificvariation, including susceptibility to imbibitional damage, are discussed.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The UK Government is withdrawing the use of some chemicalpesticides from stored product facilities. Entomopathogenic fungi(EPF) could provide one viable alternative (Wakefield et al., 2002).However, if an EPF is to be successfully used as a biological pesticidecertain requirements must be met, including sufficient levels ofpersistence and virulence in-situ. In UK storage facilities, an EPFcontrol agent must induce epizootics at the standard UK conditions,which include relatively cool temperatures and low moisture levels.Unfortunately, there is substantial evidence that the efficacy of EPF islimited by low temperature and, under some circumstances, lowwater availability (Butt et al., 2001;Moore,1972;Quintela et al.,1990).Abiotic limitations, such as temperature extremes and low moisturelevels, can be obviated by formulation, leading to EPF that are effec-tive under sub-optimal conditions (Lomer et al., 2001; Langewaldet al., 1999) and there are means of manipulating culture conditionsto change certain conidial characteristics including environmentalpersistence (Lane et al.,1991). Variation of EPFat the species and sub-species level to environmental stresses, including temperature andmoisture availability (Hallsworth and Magan, 1994; Ying and Feng,

; fax: þ44 (0) 1491 829100.n).

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2004) means that careful isolate selection can also be a useful stepin the development of a biopesticide.

This paper describes a set of experiments conducted on twoisolates of Metarhizium, M. anisopliae (Metschn.) Sorokin andM. acridum (Driver & Milner) Bisch., Rehner & Humber, to examineif culture age has an effect on conidia viability and tolerance toreduced water availability.

2. Materials and methods

Freeze dried cultures of M. anisopliae (isolate IMI 385045) andM. acridum (isolate IMI 330189) were obtained from the GeneticResource Collection of CABI, UK. Isolate IMI 385045 is an isolateoriginally commercialised as a biological pesticide (Bayer AG 1020)to control soil pests such as black vineweevil (Otiorhynchus sulcatusFabricius) (Coleoptera: Curculionidae), but the original host isunknown. Isolate IMI 330189 was originally obtained from Orni-thacris cavroisi Finot (Orthoptera: Acrididae) from Niger in 1989.

Both isolates were grown on Sabouraud Dextrose Agar (SDA) inPetri dishes (90 mm diameter). The dishes were inoculated witha 200 ml conidia suspension at a concentration of 106 conidia ml�1,sealed with parafilm and kept at 25 �C. There were 10 dishes for eachisolate. At the onset of visible conidiation (4 days post-inoculation,for both isolates), conidia were collected from the dishes by gentlybrushing the culture with a sterilised piece of electrostatic duster

C. Smith, S. Edgington / Journal of Stored Products Research 47 (2011) 157e160158

(Flash�). The purpose of the electrostatic duster was to use theopposing magnetic fields of conidia and duster to collect conidiawith minimal damage to fungal phialides. The dishes were then re-sealed and returned to 25 �C. Conidia were harvested again at 11, 18and 22 days post-inoculation, hence the age of conidia, theoretically,did not exceed 7 days. Efficiency of harvesting could not be deter-mined but it is likely that the majority of conidia were collected ateach harvest. Harvested conidia were tapped off the duster on to analuminium foil dish and dried over silica gel for 3 days. Conidia fromcultures of the same age and isolate were grouped, as this madesubsequent handling of conidia much easier. Conidia were thentransferred to a sterile glass vial, sealed andkept at 5 �Cuntil required.

Conidia germinationwas examined on SDA in Petri dishes (90mmdiameter), modified with polyethylene glycol-600 (PEG) to adjustwater activity (aw). There were five different water activities, namely(quantity of PEG l�1) 0.998 (0 g), 0.991 (123 g), 0.982 (164 g), 0.973(201 g) and 0.944 aw (474 g), with water activity assessed usinga Series 3 TE water activity meter (AquaLab.). For tests at 0.998 aw,conidia were suspended in 0.5 ml sterile water; for all other wateractivities conidiawere suspended in sterilewater adjusted to0.940 aw(800 g PEG l�1). Each dish was inoculated with 25 ml conidiasuspension, at 106e107 conidia ml �1, uniformly spread with a glassrod. The dishes were-sealed with parafilm and kept at 25 �C. Germi-nation of conidia was assessed after 24 h, under a light microscope(�400 magnification). Conidia were classified as germinated if thelength of the germ tube was longer than the length of the conidium.Approximately 200 conidia were counted on each dish. There werethree dishes per treatment. The trial was performed twice.

2.1. Data analysis

The results from the two trials were similar and were thereforecombined for each EPF isolate. Conidia germination (%) data werearcsine transformed prior to analysis to improve homogeneity (thedata presented here are pre-transformed data). One-way analysis ofvariance (ANOVA) with appropriate factors was used to investigatetreatment effects, differences between means were consideredsignificant at P < 0.05 (Genstat 12th Edition, VSNI). Actual germi-nation figures are provided in Table 1. To illustrate change in conidiagermination for different water activities, relative germination wasused, i.e., all treatments started at 100% germination (Fig. 1a and b).

3. Results

When tested on growth media of 0.998 aw, culture age hada significant effect on conidia germination of M. anisopliae andM. acridum (M. anisopliae: F ¼ 89.8, df ¼ 3,8, P < 0.05; M. acridum:F ¼ 29.7, df ¼ 3,8, P < 0.05). Germination of similarly agedM. anisopliae conidia was highest for conidia from 4 to 11 day-old

Table 1Germination (%) � SE of Metarhizium anisopliae (IMI 385045) and M. acridum (IMI 330180.998, 0.991, 0.982, 0.973 and 0.944. Germination assessments were made after 24 h at

Culture age (days) Germination % (�SE)

0.998 aw 0.991 aw

M. anisopliae4 88.1 (�3.63) 86.9 (�1.16)11 94.0 (�0.93) 75.1 (�3.32)18 40.0 (�3.00) 8.9 (�0.56)22 39.3 (�2.12) 18.2 (�2.97)

M. acridum4 64.2 (�3.24) 45.9 (�18.29)11 90.3 (�2.85) 63.8 (�1.65)18 95.4 (�1.22) 79.7 (�4.48)22 95.9 (�1.09) 86.5 (�2.74)

cultures (>88%) compared to the 18 and 22 day-old cultures (�40%),germination of M. acridumwas the lowest for the 4 day-old culturecompared to all other culture ages, 64 compared to > 90%, respec-tively. As the water activity of the growth media was reduced from0.998 to 0.944, germination of both EPF, across all culture agetreatments, was significantly reduced (M. anisopliae: F ¼ 11.5,df¼3,8,P<0.05;M.acridum: F¼37.1,df¼3,8,P<0.05).Germination(relative to 100% initial germination) of M. anisopliae had fallen byapproximately 20, 50, 95 and 100% from the 4, 11, 18 and 22 day-oldcultures, respectively, at 0.944 aw. The reduction in germination forthe 18 and 22 day-old cultures was particularly prominent from0.998 to 0.991 aw, falling by78and53%, respectively (the reduction inwater activity is equivalent to a reduction of 0.7% in the equilibriumrelative humidity of the growth media). From 0.998 to 0.944 aw,germination of M. acridum dropped by at least 88%, irrespective ofculture age. The most rapid loss was from the 4 day-old culture,which fell by approximately 71% from 0.998 to 0.982 aw, whilstgermination of all other age treatmentswas still over 61% at 0.982 aw.

4. Discussion

Selecting the most suitable isolate is critical for the success of anEPF-based biological pesticide, although the ability to alter the fitnessof the EPF, e.g., through modifications of the production process, canbe a useful additional tool. Trehalose supplements and reducedwaterlevels inproductionmedia increased the environmentalfitness of EPFconidia (Thevelein, 1984; Hallsworth and Magan, 1999). The presentstudy suggests that the age of the culture could also affect conidiaviability. There were, however, indications of interspecific variationbetween the Metarhizium isolates, with regards to the impact ofculture age on conidiafitness. Viability of theM. anisopliae isolatewasconsiderably higher when harvested from the youngest, 4 day-oldculture, compared to cultures over 17 days old; interestingly though,M.acridum showed lowerviabilitywhenharvested fromtheyoungestcultures compared to the oldest. These resultsmay reflect differencesin susceptibility to imbibitional damage between the Metarhiziumisolates; Faria et al. (2009) showed that although rapid conidiahydration (i.e., themethod in thepresent study) isdetrimental to bothM. anisopliae and M. acridum, the two Metarhizium species differ intheir susceptibility to imbibitional damage. The present studyprovides no measurements of EPF growth characteristics whilst inculture. Biochemical changes in the mycelium, e.g. carbohydrateconcentration, during phases of fungal growth, namely the lag, log,linear, stationary and death phase, can differ between phases(Gottlieb and Van Etten, 1964), and the phase times themselves maydiffer between fungal species (Meletiadis et al., 2001). It wouldtherefore be interesting to investigate possible links between thegrowth phases, resource depletion and subsequent conidia fitness forthe present EPF isolates. The origins of the two isolates differ, IMI

9) harvested from cultures of 4, 11, 18 and 22 days old and tested at water activities25 �C.

0.982 aw 0.973 aw 0.944 aw

80.1 (�2.68) 77.5 (�1.49) 71.5 (�2.64)62.8 (�0.93) 71.5 (�4.66) 45.9 (�3.88)15.1 (�1.68) 7.4 (�1.27) 2.2 (�1.01)4.5 (�3.87) 3.6 (�1.47) 0

18.9 (�1.01) 18.4 (�3.74) 7.6 (�1.54)61.9 (�20.49) 72.1 (�4.95) 3.9 (�1.67)94.8 (�0.70) 88.5 (�1.82) 0.2 (�0.13)81.3 (�8.19) 27.1 (�7.19) 5.2 (�1.87)

Fig. 1. A & B. Relative germination (%) of Metarhizium anisopliae (IMI 385045) and M. acridum (IMI 330189) harvested from cultures of 4, 11, 18 and 22 days old and tested at wateractivities 0.998, 0.991, 0.982, 0.973 and 0.944. Germination assessments were made after 24 h at 25 �C. Vertical lines represent the standard errors. A: M. anisopliae (IMI 385045);B: M. acridum (IMI 330189).

C. Smith, S. Edgington / Journal of Stored Products Research 47 (2011) 157e160 159

C. Smith, S. Edgington / Journal of Stored Products Research 47 (2011) 157e160160

330189 was found in a Sahelian grasshopper, existing in extremes ofaridityandheat inwhichgrowthof conidia is slow.On the otherhand,IMI385045wasoriginallydeveloped for insects inEuropeansoils, andis more likely to be adapted to moister conditions and more rapidcycling of generations. A number of EPF studies have indeed shownthat resource depletion and abiotic stresses, e.g., nutrient and waterloss, osmotic and thermal stress, can lead to increased levels ofendogenous compounds such as trehalose, glycerol and erythritol,which allow metabolic activity to remain unaffected and as a resultenhance EPF fitness (Davis et al., 2000; Crowe et al.,1984; Hallsworthand Magan, 1994). An hypothesis that as the culture ages, nutrientsandmoisture are lost through evaporation and consumption, placingthe culture under increased stress and resulting in more drought-tolerant conidia, does fit to some extent with M. acridum in thepresent study, namely, the prolonged viability of conidia from oldercompared to younger cultures. However, this would not explain theeffect seen with M. anisopliae, if the same relationship betweenculture/media age and resource levels are assumed here, loss ofresources would appear to lower drought tolerance.

Studies will continue into the most suitable EPF isolate and thebest production process, for utilising a biological pesticide underUK storage facility conditions.

Acknowledgements

We thank Dave Moore for reviewing the manuscript. This studywas possible through on-going activities at CABI Europe-UK, sup-ported by the UK Government Sustainable Arable Link Programme,the Department for Environment, Food and Rural Affairs, theHome-Grown Cereals Authority and five industrial partners.

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