TITLE:
ESTIMATESOFGENETICPARAMETERSRELATEDTOCHITINASE
PRODUCTIONBYTHEENTOMOPATHOGENICFUNGUSMetarhizium anisopliae
AUTHOR:
GilbertoU.L.Braga
RolandVencovsky
ClaudioLMessias
ESTIMATES OF GENETIC PARAMETERS RELATED TO CHITINASE PRODUCTION BY THE ENTOMOPATHOGENIC FUNGUS Metarhizium anisopliae
Gilberto U. L. Bragal » Roland Vencovsky
!, and Claudio L. Messias'
Departamento de Genética e Univer-idade Estadual de Campinas.
Campinas, SP - Brasil
Telephone # (0192) 39 7811
Fax # (0192) 39 3124
Departamento de Genética, ESALQ/USP.
Piracicaba, SP - Brasil
Telephone %* (0184) 29 4125
Fax & (0194) 22 3087
Running title: Genetic Parameters
Evolução, CP 6109, of Chitinases in M.
Instituto de Biologia, CEP 13.083-970,
CP 83, CEP 13.400-8970, anisopliae
ABSTRACT
Chitinolytic activity was determined in culture filtrates from 17 Metarhizium anisopliae isolates grown in liquid medium containing chitin as the only carbon source. The objectives were to estimate parameters such as genetic variance among strains, heritability and expected gain from selection. Wide genotypic variability was observed among strains in chitinolytic activity, permitting the exploitation of this property in breeding. The high heritabilities permit the expectation of great progress through phenotypic selection. One of the isolates was also investigated for variation in the trait as a function of culture growth time. The results showed a growing increase in enzyme activity up to the 8th (and last) day of the experiment.
INTRODUCTION
Enzymes capable of hydrolyzing chitins produced by entomonathogenic fungi have been extensively studied in terms of: a) characterization and mode of action (Smith et a/., 1981; St. Leger et ál-. 1991, 1993); b) regulatory mechanisms (Smith and C;rula. 1983; St. Leger et al., 1986a; Havukkala et a/., 1993; El-Sayed et a/., 1993a; El-Sayed et al., 1993b; El-Sayed et al., 1993c); c) interactions with substrates (St. Leger et al., 1986b); d) correlation between enzymatic activity ll-vol and degree of virulence of various i{solates (Leite, 1987; Samuels et al., 1989; ElSayed et al., 1989; Gupta et al/., 1994); e) production kinetics in
culture (St. Leger et al., 1986¢c).
The intraspecific variability of chitinolytic activity among Metarhizium anisopliae strains has been reported by several investigators (Rosato et al., 1981; St. Leger et al/., 1986c; Leite, 1987; Samuels et al., 1989). However, none of these studies has used partitioning of phenotypic variance, so that parameters such as genetic variance, heritability or expected gain from selection for genetic improvement of the character were not calculated. Thus, the objective of the present study was to estimate these gqnetic parameters using a quantitative approach, in the belief that such estimates could be used in breeding programs which take into account, among other traits, the level of production of enzymes capable of hydrolyzing chitin. This approach is based on the existence of evidence showing that this character is associated, at least in part, with the degree of virulence of other fungal species such as Nomuraea rileyi (El-Sayed et al., 1989), Verticillium lecanii (Jackson et al., 1985) and Beauverfa bassiana (Gupta et al., 1994).
MATERIALF AND METHODS
Meotarhizium anisopliae strains, origin and maintenance
The 17 M. anisopliae var. anisopliae strains (Table |) were obtained from the germplasm bank of the laboratory of entomopathogenic fungi, State University of Campinas. Spores were obtained by growing the isolates on minimal medium (MM) (Pontecorvo et al., 1953) at 28°C for 12 days.
Determination of viability
Conidial viability was determined by {inoculating a suspension containing 10% conidia/ml onto plates containing MM. After 14 h of incubation at 28°C, a random sample of 500 conidia was examined for the percentage of germinating and non-germinating conidia.
Filtrate preparation and determination of its enzyme activity
Fifty ml Erlenmeyer flasks containing 17 ml liquid MM with 1% chitin (w/v) Treplacing glucose were autoclaved at 110°C for 20 min and inoculated with 3 ml of the suspensions containing 3x107 conidia ll-l and grown at 28°C with shaking at 150 rpm for varying periods of time. The chitin used (from crab shells, practical grade, Sigma) was sifted through a 0.149 mm mesh and only the fraction that passed through was utilized.
After the growth period, the content of each flask was filtered through previously tared Inlab type 10 paper to retain the mycelial mass produced. The collected filtrate was divided into one ml aliquots and stored frozen at -60°C. For one of the isolates (strain 58), the filtrate was collected between the 3nd and 8th day of growth. The filtrate of all isolates after 7 days of growth was used for comparison of the enzyme activities.
Chitinolytic activity
The Chitinolytic activity of the filtrates was determined as follows: one ml of a chitin azure (Sigma) suspension, 2 mg nl'l in Mcllvaine's citric acid-phosphate buffer (pH 5.2) was added to each tube containing one ml of the filtrate thawed in a water bath at 37°C. The mixture was incubated at 37°C with shaking at 200 rpm. After 10 h, the content of the tube was centrifugated at 3000 g (r 7 cm) for 10 min and a 100 ul aliquot of the supernatant was submitted for determination of
absorbance at 575 nm. The precipitate was resuspended and incubation was continued for 12 additional h, after which the previous procedure was repeated.
Absorbance was determined with a DU 70 Beckman spectrophotoa an 8 mm high micro cell (Beckman). Enzyme activity is reported as the variation in supernatant absorbance during the incubation periods (10 and 22 h).
Experimental design and statistical-genetic analysis
For better environmental control, strains were evaluated in two separate experiments containing 8 and 9 treatments, respectively. The design was completely randomized with three replications (R). A common check treatment (strain 58) was included in each experiment for evaluation of between experiments environmental differences and adjustment of treatment means (Pimentel Gomes and Guimar¥es, 1968). Being one of the genotypes under evaluation strain 58 was {ncluded to contribute to the among strains genetic variability in one of the experiments (Exp. 2)\./The scheme of the combined analysis of variance is presented in Table 1l. For estimation of covariances and correlations the analyses of variance scheme was applied to the sum of the variables on a plot basis, as proposed by Kempthorne (1966), Whithin group variance components were estimated as follows:
a) genetic variance: (º,,) = (MS,-MS5,/R
b) variance due to the experimental error:
¢) phenotypic variance among strain means:
(9)) = N3,
(0,) = MS,/R
To assess the correlation between traits, the fol lowing coefficients vere estimated:
2) genotypic correlation among strains: (fQ = n o Ve
(547
) environmental correlation: (f£;) = Hn
c) phenotypic correlation: (£,) = Vot * Vs
Estimate of error variance of any adjusted treatment mean, with C common treatments and S experiments was estimated through: 0 = En dd)
In the present case S = 2 and C = 1 For comparison of adjusted treatment means the following error variances were estimated:
a) of contrast between two strain means within experiments: (90,) = 2M5,/R
b) of contrast between two strain means of different experiments: NSy 1 (%) = 27 (l z.)
Estimates of additional parameters
The following additional parameters were estimated: 9,
a) heritability on a plot basis: (5 )- 0#; and on a strain
mean basis: (5:) = o ' P 9,
b) expected gain from direct selection: (&) = KL with an average
K=0.96 (with small samples, k=0.91 for selection of 3 out of 8 strains (3/8) and k=1.00 for selection of 3 out of 9 strains (3/9) (Fisher and Yates, 1966))
c) expected gain from selection as percentage of the trait mean: (68) = %'100 were Y, is the initial overall mean for the character.
Spearman's rank correlation
Since the chitinolytic activity of the isolates was determined using two different periods of incubation with the substrate, Spearman's coefficient of correlation was calculated to determine the coincidence
of results obtained (Steel and Torrie, 1960), for strain means betw periods.
RESULTS
Kinetics of Chitinase Production
Fig. 1 shows the variation in chitinolytic activity observed in the culture filtrate of strain 58 as a function of time of growth. Activity increased up to the B8th and last day of the study. In similar experiments, St. Leger et a/. (1986c) obtained similar results for the enzymes N-Acetyl-B8-glucosaminidase (EC 3.2.1.30) and chitinase (EC 3.2.1.14).
Viability
Viability of isolates was high and ranged from 95.0% to 98.6%.
Means and Dispersion of the Trait Evaluated
Table 11l shows adjusted strain means of chitinolytic activity (A, B). Wide intraspecific variability in chitinase production was detected, with the highest activity being approximately 5 times larger than the smallest value when activity was detemined after 12 h of incubation, and 6.5 times larger when activity was determined after 22 h. Similarly, estimated genetic variance was 3,7 times larger when activity was determined after 22 h of incubation (Table [V). Spearman's correlation coefficient between the two determinations was 0.958 (for a t value ot 12.94%), 7 qm
Analysis of Variance
Results of the analysis of variance of the data, with mean squares, coefticients of variation and significance of F test are given in Table IV, Results indicate significant genetic variation among strains for the character.
Estimates of phenotypic variance and its components are shown in Table V. Table VI shows heritability estimates (A2 and A?) and estimates of expected gain from selection (& and 8,8). These values show that chitinolytic activities have a strong genetic determination. Selection among strains should therefore result in considerable improvement. In fact, on the basis of 12 h incubation time selected strains are expected to be 44.19% superior to the average performance of the sample of 17 strains. After 22 h of incubation, selection should be more efficient with an expected gain of 45.68%.
DISCUSSION
The study carried out by St. Leger et a/. (1986a) on the regulation of chitinolytic enzymes in M. anisop/iae showed that the most efficient inductors for chitinase and chitosanase are N-acetylglucosamine and glucosamine. These two monomers are released when culture media containing chitin are autoclaved, and have been previously identified as inductors of chitinase synthesis in Beauveria bassiana by Smith and Grula (1983) .,
The purpose of the culture medium, containing 1% (w/v) chitin and sterilized by autoclaving, was to induce chitinolytic activity and the results obtained were valid, in principle, for these growth conditions. Obviously, these conditions differ from those occuring fn vivo during the
initial stages of infection when chitinase production may be of benefit to the virulence of the isolates.
The quantitative study of proteolytic (Braga et al., 1994) and chitinolytic activity here investigated, together with estimates of the genetic parameters of such activities, provides information for programs aiming at the improvement of these traits. It should be remembered, however, that these characters are simply components of a complex phenotypic characteristic which is the virulence presented by a given isolate against a host population under specific environmental conditions. It should be pointed out that, in the case of entomopathogenic fungi, the final objective of an improvement program could be an increase in virulence. One way to achieve this is by improving the characters related to virulence.
The methodology utilized in the determination of chitinolytic activity using chitin azure as substrate, followed by the determination of supernatant absorbance, does not permit an individual analysis of all enzymes involved in the process of chitin hydrolysis. Thus, the method evaluates the Jjoint action of the enzymes of the chitinolytic complex produced by each isolate. According to Hackman and Goldberg (1964), the advantage of using chitin azure is the fact that this substrate is not partially hydrolyzed. Substrates such as colloidal chitins, by undergoing partial degradation during preparation, may be hydrolyzed by enzymes that do not have the ability to hidrolyse this substrate in the naturally occurring form.
The wide variability in chitinase production detected among isolates shows the adequacy of the methodology used here for this type of study. The variability in chtinolytic activity present in M. anisopl/iae cul ture filtrates had been previously detected by St. Leger et al/. (1986c). With respect to chitinases, experiments in which activity is measured by determining the extension of the zone of substrate degradation around
colonies grown on appropriate media (Gabriel, 1968 ; Hankin and Anagnostakis, 1975) appear to be unable to detect the existing range of variation (see Samuels et al., 1989).
The wide variability detected here may be attributed to the great genetic heterogeneity among isolates, since these isolates were not submitted to any previous process of selection for the character. Estimates of genotypic variances were the major components of the estimates of phenotypic variances, leading to considerably high heritabilities. The high Ã.ª value for chitinolytic activity indicates that the population can be easily improved for the trait, with the achievement of significant gains by simple phenotypic selection. Although Spearman's correlation coefficient was considerably high, the lower coefficient of variation and higher Ó,l obtained when the chitinolytic activity of the filtrates was evaluated after 22 h of incubation with the substrate, indicate that strains could be selected using this period of incubation.
Ve believe that this type of genetic analysis of characters involved in processes of host infection and colonization may be helpful in the development of programs aiming at selecting of more efficient strains for biological control.
ACKNOWLEDGEMENTS
This work was supported in part by the following Brazilian organizations: CNPq, FAEP, PADCT-F INEP,CNPq-Blue Ribbon.
Foi determinada a atividade quitinolftica apresentada pelos filtrados de culturas de diversos isolados de Metarhiziuam anisopliae crescidos em meio liquido, tendo quitina como Unica fonte de cabono. Os _nbjauvru foram a estimativa de parametros como a variancia genética, a herdabilidade e o ganho esperado na seleg¥o. Uma grande variabil idade genotipica foi verificada na atividade quitinolitica, permitindo sua exploração no melhoramento. Os altos coeficientes de herdabil idade permitem esperar um grande progresso na seleção fenotipica. Para uma das linhagens foi também determinada a variação na atividade enzimatica, em função do tempo de crescimento das cul turas. Os resul tados mostraram um aumento na atividade enzimatica até o oftavo dia da avaliação.
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Origins of Metarhizium anisopl/iae var. anisopliae strains
: putative diploid of strain E. (Messias and Azevedo, 1980).
Scheme of combined within experiments analysis of variance and covariance with expected values of mean squares (MS) and mean products (MP)
TABLE 111
Adjusted means of the 17 Metarhizium anisopliae strains for chitinolytic activity., Error of any adjusted treatment means M,. and error of difference between two treatment means: m (same group); m (different groups). Chitinolytic
! variation in the absorbance at 575 mm after 10 h (A) and 22 h (B) of incubation with the substrate.
? (58) unadjusted means of standard strain.
Ranking of strain means.
* Includes adjusted means of strain 58 in Exp. 2.
TABLE IV
Mean square values of the analysis of variance for the characters and corresponding siginificance of F tests; coefficients of variation.
º Variation in the absorbance at 575 mm after 10 h (A) and 22 h (B) of incubation with the substra
H gSigniticant at the 1% level.
TABLE VI
Estimates of expected gain from selection and heritability.
º yariation in the absorbance at 575 mm after 10 h (A) and 22 h (B) of incubation with the substrate.
TABLE V
Estimated phenotypic variances between strain means (¥,), genotypic variances (), and variances due to experimental error ( p/r).
Variation ín the absorbance at 575 ma after 10 h of tncubation with the substrate.
Variation in the absorbance at 575 ma after 22 h of inoubation with the substrate.
Absorbance after 10 h of incubation with
Absorbance after 22 h of incubation with the substrate the substrate
