CHARACTERIZATION OF NEW BIOTYPES OF P157 STRAIN OF Metarhizium anisopliae var. anisopliae, GOT BY TREATMENT WITH GAMMA RADIATION

Conidios de la cepa silvestre P 157 de Metarhizium anisopliae var.  anisopliae (Metsch.) Sorokin, fueron expuestas a la radiacion gama para la obtencion de nuevos biotipos. En la dosis de 390Gy se obtuvo 36 colonias (MaP). Cinco colonias (MaP 03, MaP 17, MaP 25, MaP 27), expresaron alteracion morfologica para el color en el Medio Completo. En este, 21 colonias presentaron crecimiento micelial significativamente distinto del testigo silvestre, despues de 12 dias de la inoculacion. La colonia MaP 25, presento mayor valor de crecimiento micelial y la colonia MaP 11, el menor valor. Entre las colonias obtenidas, solamente la colonia MaP 28 presento auxotrofia, identificada como una deficiencia en la sintesis del acido paraminobenzoico. Los conidios de 29 colonias, presentaron un promedio de porcentaje de germinacion significativamente distinto del respectivo aislamiento silvestre, despues de 12 horas de incubacion en Medio Minimo, a 25°C. Las colonias MaP 02, MaP 21 y MaP 36, presentaron precocidad en la germinacion de los conidios en relacion al patron silvestre.


INTRODUCTION
Some fungi reveal great possibiliLies ofbeing used in insect and pest biological control programllles beca use they have an enlomopathogenic aclion.Among these, M. al1isopliue is one ofLhe most studied as regareis the pathogenicily and virulence on more than 200 species of i nsecls,
In Brazil, M.al1isop!iae has been lIsed very sllccessfllIly in the biologic control of sllgar-cime cicadas (Mahanarva posticata Stal., Ma¡'anarvafimbriolata Stal .) and those of pastures Deois flavopicta Stal.and Zulia entreriana Ber.g.This fungus is also used in the control of Diatraea sacc¡'aralis Fabr, Hypot¡'enemus Iwmppei Ferrar, Tihraca limhativentris Stal.and Cornitermes cumQlans Kollar(2,7,13 ) 4).The efficiency of entomopathogenic fungi used in biological control, can be improved by means of genetic manipulation, by altering, among other things, factors Iike spomlation, dispersion and tolerance of the spores to stress (36).A considerable increase in the infectivityof a wild strain of M. al1isopliae was obtained by Yokoyama et al. (37) aftcr treatment of protoplastswith UVlight.Tsai etal.( 36) isolatedsamples of M. anisopliae resistant to Benomyl and Carbendazim after treatment of conidia with UV. c1lélracteristics which are desirable in integrated past conl rol programmes.The identification ofthe traces ",hich determine pathogenicity and vimlence is a fundamental step for the improvement ofthese fungi.Riba et al. (26), point out that thc vimlence of a strain seems to be the resuIt of the manifestation of different genes.Among the various factors related to "imlence are the speed in germination of the conidia, production of extra-cellular enzymes related to penetration (lipa ses, proteases and qllitinases) and productionof toxins (1 , 6, 23 .29. 31.32).The speed in germination of the conidia and the fOJ'lnation of appressoria are crucial events in patllOgenesis (9).Resistance to high tel11peratures and UV radiation, pR luminosity, nutritional sources, colony and stock age are factors which act on the viability and speed gennination ofthe conidia ofentomopathogenic fungi (8. 11. 15, 18, 30).The objectives ofthis study ",ere the produclion of ne", biotypes of M. al1isopliae var.anisopliae using gamma radiation , through tile analysis ofmycelial gro",th, auxotrophy, conidia gennination, and morphological and physiological charactcri zation.

MATERIALS ANO METHOOS
The P 157 strain of M. 1I11isopliae var.anisopliae used was granted by the culture collection (Micoteca -URM), of the Department of Mycology of the Fcderal University ofPernambuco, Brazil ; this strain was isolated from Ma¡'al1arva posticata StaL and maintaincd in solid Complete Medium (MC) (24), 1110dified by Azevedo and Costa (5).
Conidia from colonies of 8-day old 1110nosporic cultures (12), were separately suspended in 3ml ofTwccn 80 (0, 1% v/v) solution and disaggregated in a vortex agitator.Appropriate (1 : 10) dilutiollS ",cre made and distributed to five test tubes, each containing 5ml of suspension with 10 6 conidia/ml, estimated quantitatively in a Neubauer Chamber.The suspellsions werc subl11ittcd to different doses of gamma radiation Cobalt 60 type 42 GAMMABEAN 650 -Atomic Energy of Canada, (50, 100, 150 •and 200 Gy) .One of the suspensions not treated with radiation \Vas used as a control.After this procedure, the suspensions were proportionally diluted (1: 10) in a saline solution (0,85%v/v) up to a concentration of 10 2 conidia/mI.They were placed in triplicates on Petri dishes containing solid MC and O, lml of these suspensions was spread with a Drigalsky ring and then incubated at 25°C for 48 hours.After the incubation period, the surviving colonies were counted, taking into consideration the .number of colonies obtained in the control as a 100% survival.Data obtained served as a basis for constmcting a survival curve and for calculating the dose necessary to obtain 5% survival by means of linear regression.
A suspension of conidia with 10 6 conidia/ml \Vas submitted to adose ofgamma radiation of390 Gy, aiming at a survival rate Iesser than 5%.The suspension was diluted proportionaIly (1:10) to a concentration of 10 2 conidia/ml and aliquots ofthis suspension ",ere placed on 10 Petri dishes containing solid Me.The dishes were incubated for 48 hours at 25°e.The colonies arising were transferred to test tu bes containing solid MC and incubated at room temperature and luminosity for 8 days.These colonies ",ere designated as MaP.
The micromorphologic characters ,"vere analyzed by means of smearing conidia separately on Petri dishes containing solid MC at three different points and covering them \Vitll cover glass.The dishes were incubated for five days at 25°e.The cover glass \Vas removed from tlle cultures and placed on slides with a drop of Amann blue, and observed under light microscope.
For the analysis of the macroscopic characters, 7 day-old l11ycelium discs frOI11 the MaP colonies, cut with the aid of a 5mm diameter cork drill, \Vere transferred separately to the center ofthe Pe tri dishes containing solid MC, in triplica tes, with the help of a platinum ring, and then, incubated at 25"C.Gro\\1h \Vas accompanied for a period of 12 days \Vith observation intervals on the 6th, 9th and 12th days.In this process the colour and diameter parameters of the colonies ",ere considered.
The auxotrophy of the MaP colonies \Vas verified by means of inoclllating conidia in Petri dishes containing solid Minimum Medilll11 (MM) (24), and incllbated at 25 °C for 12 days.The dishes were marked in four diameters of 2.25cl11, to estimate the percentage growth.At the end of this pcriod, conidia which had not formed colonies, \Vere transferred to Petri dishes containing solid MM to which was added, separately, hydrolyzed casein, a sollltion ofyeast nucleic acid, and a sollltion ofvitamins, in order to gain specific knowlcdge of the nutritional deficiency.
A prior synchronization of conidia (lO) was undertaken to analyze germination, thereby obtaining conidia of lhe same age and physiological slate.Conidia of lhe MaP and ",ild PI57 colonies, arising from the synchronization process were pul in test lubes containing 9ml of liquid MM in a concentration of I OC' conidia/ml.Aliquots of the medium were with drawn at intervals of one houL and observed under lhe light microscope.Thc percentage of germination \Vas calclllated by observing after 12 h lhe presence or absence o[ germination lubes on 500 conidia using a Neuballer Chamber.
Turkcy 's tesl \Vas employed to compare the mean gro\\1h of the diffcrcnt colonies.

RESUL TS AND DISCUSSION
The results obtained as lo the survival in gamma radialion, of conidia of the P 157 slrain of M, al1isopliae var.allisopliae, ",ere used lo construct o a survival curve (Figure 1).The exlrapolated dose lo gel aSo;;) survival was 390 Gy .... To analyze the germination percentage the MaP colonies that showcd .an aYerage ofm)'celial diameter of a significantly greater value than that of the wild strain, in addition to the some colonics having an average diallleter of lesser yalue were chosen.The germination percentage ofthe analyzed colonies is to be found in Table 3.Among the tested colonies,17 sho",ed an average germination percentage significantly minor from lhe ,úld slrain (Table 4).Okino et a1.(19), ",orking with M. al1isopliae and two species of Aspergilllls, observcd after cxposure of conidia to gamma radiation, a survival of approximatcly 1% in lhe dosc of348 Gy for M. allisopliae, and reportcd a grcal scnsibility ofthis fungus to radiation, that \Vas also obseryed in this study.
Luna-Alvcs Lima (12).points out that differcnt chemicéll and physical agents are used in the induction of lllutation in organisms, ",hile gamma radiation is little used as él Illutagenic agent in enlomopathogenic fungi .One ofthe mosl " 'idely used physical agents for induction of mutation in' filamentous [ungi is ultraviolet light (14, 33.37).In the improvement programmes [or fungi used in biolocal control , ultraYiolet light is also used more frequcntly due lo the ease of manipula tion .Azcvedo (4).points that gamma radiation can bc considercd as an exccllent mutagenic induclor in some cases.
One ofthe delenni ning factors in the palhogenicity of entomopathogenic fungi is the germinalion of conidia, this being the first slep for host infection (16).Samuels el al. (28), staled that paramelers like germinalion speed and groWlh are correlated with the high virulence of some strains o[ M. anisoplille for Nilllparvata [l/gens.The age oflhe colon)' can be considered as an interaction faclor in a conidial gcnnination.Hall el al. (8) , in a comparative analysis of different species of enlomopalhogenic [ungi claimlhat conidia coming from )'oungcultures show more rapid genninalion lhan lhose ofold cultures.In this study the synchronizalion oflheconidia fromlhe wild slrain and [rom the MaP colonies was reached in such a way that lhe conidia used in lhe genninalion Irial possessed lhe same physilogical condilions, lhus climinaling varialion due to lhe age of Ihe conidia.
It was obseryed lhat Ihe MaP 15, MaP 21 , MaP 33 and MaP 36 colony, showedaccordingly a high germinalion percentage, good growlh in MM and Me and in addilion lo lhis, MaP 21 began germinalion ",ell in advance.Considering Ihat characlerislics Iike high percenlage and speed of germination.apart from good mycclial groWlh, are desirablc in entomopathogenic fungi , the colonies which showed such characteristics may be promising for use in genelic crossing aiming the inc¿rporation ófthese ClléHacleristics inlo strains wilh recognized good performance in biological control.
The biolypes obtained in this study need furlher studies lo analyze olher changes arisi ng [ro m the cxposure lo gamma radial ion ",hich may ",ell contribute lo lhe knowledgc of lhe genetic bases associaled wilh lhe mosl appropriale phcnolypcs for biological conlrol prograllllllcs.