Limits to the Negative Logarithmic Relationship Between Moisture Content and Longevity in Conidia of Metarhizium flavoviride

Transcription

1 Annals of Botany 81: , 1998 Limits to the Negative Logarithmic Relationship Between Moisture Content and Longevity in Conidia of Metarhizium flavoviride T. D. HONG*, N. E. JENKINS, R. H. ELLIS* and D. MOORE * Department of Agriculture, The Uni ersity of Reading, Earley Gate, P.O. Box 236, Reading RG6 6AT, UK and International Institute of Biological Control, Silwood Park, Buckhurst Road, Ascot, Berks, SL5 7TA, UK Received: 7 November 1997 Accepted: 27 January 1998 Conidia of Metarhizium fla o iride were hermetically stored at 5 C and 14 moisture contents between 2 5 and 31 8% (fresh weight basis) for up to 146 d, and tested for germination on Sabouraud Dextrose Agar at 25 C for 24 h. Survival of conidia conformed to cumulative negative normal distributions and all 14 survival curves could be constrained to a common origin. There was a negative logarithmic relation between longevity and conidia moisture content, but limits to the relation were detected: the lower-moisture-content limit was 4 6% [in equilibrium with 1 7% relative humidity (RH) at 2 C], below which value further reduction in moisture content did not increase conidia longevity; and an upper-moisture-content limit between about 21 2 and 31 8% moisture content (between 77 and 9 % equilibrium RH at 2 C) above which conidia longevity no longer decreased. The observations could also be described by a negative semi-logarithmic relation between conidia longevity and equilibrium relative humidity. In this model, each reduction in equilibrium relative humidity by 11 2% within the range 1 7 to 8% RH doubled conidia longevity. The similarities in these relations, and the limits to these relations, between the conidia of this entomopathogenic fungus and the orthodox seeds of higher plants are discussed Annals of Botany Company Key words: Conidia longevity, equilibrium relative humidity, Metarhizium fla o iride, moisture content, hermetic storage, viability equation. INTRODUCTION We have recently developed a model of the effects of storage duration and environment on fungal spore survival (Hong, Ellis and Moore, 1997). Two different approaches were used to describe loss in spore viability, assessed by the percentage of spores able to germinate (G), as a function of storage period ( p, d). These were: (1) negative exponentials log G a bp (1) where a is the intercept, and b is the gradient of the regression line; and (2) negative cumulative normal distributions probit G a p σ (2) where σ is the standard deviation of the frequency distribution of spore deaths in time (d), and a is the intercept. In practice the latter approach was preferable, simply because the intercept values fitted agreed much more closely with initial estimates of percentage germination. Relations between spore longevity (σ) and storage temperature (t, C) and moisture content [m, % fresh weight basis (w.b.)] were described by the equation log σ K E C W log m C H t C Q t (3) where K E, C W, C H, and C Q are constants. An alternative approach was provided by a negative semi-logarithmic For correspondence. Fax 44 () , r.h.ellis reading.ac.uk relation between spore longevity and equilibrium relative humidity (r, %), i.e. log σ K E C R r C H t C Q t (4) where C R is a constant. These models were applied successfully to previously published data for spores of five species of entomopathogenic fungi and four species of phytopathogenic fungi. However, with respect to the relations between moisture and longevity, the subject of the current work, estimates of C R and the lower- and upper-moisture-content limits to the relation described by eqns (3) and (4) varied considerably not only among but also within species: the lowerequilibrium-relative-humidity limits varied between and 3% RH, the upper-equilibrium-relative humidity limits varied between 35 and 8% RH and estimates of C R varied between 16 and 25 (Hong et al., 1997). One potential cause of such variation may have been the diverse experimental techniques used by the different research groups which provided the original data (Hong et al., 1997), because in other anhydrous systems many similarities have been detected. In particular, in orthodox seeds (Roberts, 1973) the lower- and upper-moisture-content limits are close to about 1 and 9% RH, respectively (Roberts and Ellis, 1989), while estimates of C R have been close to 346 (Ellis, Hong and Roberts, 1988, 1989, 199a, b; Roberts and Ellis, 1989). The objective of this investigation was to determine in a single investigation the lower- and upper-moisture-content (and relative humidity) limits to the negative relations $25. bo Annals of Botany Company

2 626 Hong et al. Longe ity of Fungal Conidia between longevity and conidia moisture content (and equilibrium relative humidity) in air-dry storage and compare these estimates, together with that of C R, in order to discover whether or not these estimates are similar to those in seeds, and thus the extent of the economy of nature in anhydrous biology. MATERIALS AND METHODS Metarhizium fla o iride W. Gams & J. Rozsypal, isolate IMI 33189, was grown in a two-stage system as described by Jenkins et al. (1998). Fungal biomass was produced in a liquid culture system and subsequently transferred to cooked, sterile rice, on which the fungus sporulated. Once the sporulation process was complete, the rice and conidia were spread out onto plastic trays and allowed to dry to approx. 2% moisture content. The conidia were then separated from the rice by sieving through a 3 µm mesh. The resulting conidia powder was then re-sieved through a 16 µm mesh to remove any remaining rice dust particles. After extraction, the conidia had 17 7% moisture content (w.b.). They were then sealed hermetically in a laminated aluminium foil packet. Conidia moisture content was adjusted from 17 7% before experimental storage to 14 different values between 2 5 and 31 8% (w.b.), either by drying below or by humidification above the initial value. Drying to 6% moisture content took place in a forced-air drying cabinet maintained at 1 12% RH and 17 2 C. In order to dry conidia further, they were then placed in a desiccator above regularly-regenerated silica gel at 2 C. Drying conidia in a drying cabinet with approx. 15 g of conidia in a 1 cm-deep layer reduced moisture content from 17 7 to6%in6h. Further drying to 2 5% moisture content using silica gel at 2 C took 3 d. In order to increase moisture content, conidia were placed above water in a desiccator at 2 C. Humidification to 31 8% moisture content, the highest value, took 24 h. After equilibrating for 1 3 d at 3 5 C in sealed laminated aluminium foil packets, conidia moisture contents (w.b.) were determined. Two 1 g samples of conidia, at each moisture content, were dried in a mechanicallyventilated oven at 13 2 C for 17 h. The equilibrium relative humidities of the conidia were determined at 2 C using a Novasina Humidat IC1 (Zu rich), previously calibrated at 11, 55 and 9% RH. Eleven sub-samples of conidia, each of 4 g, at each moisture content were hermetically sealed in laminated aluminium foil packets (1 15 mm). These were stored in an incubator maintained at 5 5 C and samples removed from storage at intervals varying from 6 h to 14 d, depending upon moisture content, for periods up to 146 d. All samples including controls (not stored at 5 C) for each moisture content were then tested for ability to germinate. Dry conidia were placed in a 35 mm-diameter Petri dish, and floated over water in a closed container for at least 3 min in order to re-humidify to approx. 15% moisture content before these tests in order to avoid imbibition damage (Moore, Langewald and Obognon, 1997). Conidia absorbed moisture rapidly from the air. For example, a 3 g-sample 1 mm deep increased in moisture content from 5 to 15% in only 3 min. The conidia were then suspended in 1 ml of Shellsol T (Alcohols Ltd, Bishops Stortford, UK) in a universal bottle (25 ml) and shaken thoroughly. This suspension was then diluted in Shellsol T to obtain a concentration of approx. 1 1 conidia ml. The diluted samples were then treated in a bath sonicator (Branson 22, Connecticut, USA) for 6 s to break up any conidial chains. A small drop of the sonicated suspension was transferred evenly onto the surface of Sabouraud Dextrose Agar (SDA) (Oxoid Unipath, Basingstoke, UK) in 5 mm-diameter Petri dishes. Inoculated Petri dishes were placed in an incubator maintained at 25 C for 24 h. Germination of conidia was then assessed using a Nikon Alphaphot 2 compound microscope with bright field illumination at 2 magnification. At least 3 conidia were assessed on each Petri dish, three replicate dishes were prepared for each sample. Conidia were considered to have germinated if the germ tube was equal to or greater than the diameter of the conidia. Conidia survival curves were constructed as both negative exponential relations and negative cumulative normal distributions using regression analysis (after transforming germination percentage to logarithms) or probit analysis using GLIM (Baker and Nelder, 1978), respectively. The estimates of σ provided by probit analysis were then subjected to regression analysis in accordance with the equations log σ K C W log m (5) log σ K C R r (6) which are modifications of eqns (3) and (4) and describe relations between conidia longevity and moisture at a single temperature. RESULTS AND DISCUSSION Relations between conidia moisture content and equilibrium relati e humidity The relation between the moisture content and the equilibrium relative humidity of conidia of M. fla o iride was sigmoidal (Fig. 1). Conidial sur i al cur es Survival data at 14 different storage moisture contents are presented in Fig. 2. In accordance with eqns (1) and (2), the 14 survival curves were fitted as negative exponentials and negative cumulative normal distributions, respectively, to a total of 394 observations. Constraining all 14 conidia survival curves (Fig. 2) described by negative exponentials to a common origin provided a significant increase in residual deviance (P 1), the coefficient of determination being reduced from 87 to 85. The value of this common intercept was (s.e. 22) and is equivalent to 136 1% initial germination. Similarly, constraining all 14 conidia survival curves fitted by probit analysis to a common origin also provided a significant increase in residual deviance (P 1), the coefficient of determination being reduced from 93 to 92. The value of this common intercept was (s.e. 4) and is

3 35 Hong et al. Longe ity of Fungal Conidia 627 estimates of σ provided by a common origin to all 14 survival curves were used in the subsequent analyses. Moisture content (%, w.b.) Equilibrium relative humidity (%) 1 FIG. 1. Relations between conidia moisture content (%, fresh weight basis) and equilibrium relative humidity (%) determined at 2 C in Metarhizium fla o iride. The curves represent absorption and desorption isotherms above and below 17 7% moisture content, respectively. equivalent to 89 7% initial germination. While neither approach was entirely successful in describing all 14 conidia survival curves [in part because some observations such as those at 1 3% moisture content (Fig. 2F) provided almost linear responses of ability to germinate to duration of storage], eqn (2) was preferable to eqn (1) for most survival curves as well as having the higher coefficient of determination and more realistic initial estimates for germination (i.e. 1%). An example of the differences between the two approaches to describe conidia survival is provided in Fig. 3. This shows the survival curves of the conidia stored at 13 8% moisture content with 93 1% initial germination (Fig. 2D) fitted by negative exponentials and negative cumulative normal distributions with (Fig. 3A) and without (Fig. 3B) a common origin to all 14 survival curves. The survival curves provided by negative exponentials and negative cumulative normal distributions without a common origin had coefficients of determination of 94 and 98, respectively. Moreover, the estimates of initial germination were and 92 8%, respectively. The latter value is close to the actual value (93 1%). Thus, negative cumulative normal distributions provide a more suitable approach to describing survival during air-dry storage of conidia of M. fla o iride than do negative exponentials. This contrasts with the view that the survival curves of most fungal and bacterial spores conform to negative exponentials (Roberts, 1972 a; Henis, Kenneth and Barash, 1987). In this respect, conidia of M. fla o iride behave like seeds (Roberts, 1972a, b). Although variation in the estimates of the intercepts for the 14 survival curves was statistically significant, in practice the effect of the constraint provided by a common origin on the estimates of σ was negligible. Consequently, the Relations between longe ity and moisture content The estimates of σ for storage at each conidia moisture content provided by probit analysis are shown in Fig. 4. In Fig. 4A, both axes (longevity and conidia moisture content) have logarithmic scales. In accordance with eqn (5), between 5 3 and 21 2% moisture content there was a negative logarithmic relation between longevity and moisture content (P 5). Discontinuities are evident at lower and possibly also at higher moisture contents however (Fig. 4A). Estimates of the lower-moisture-content limit and the upper-moisture-content limit to the relations described by eqn (5) were determined as follows: the results at all 14 moisture contents were first analysed within a single data set in accordance with eqn (5) by linear regression analysis. The data was subsequently analysed in two separate sets iteratively with progressively more estimates of σ being apportioned to the lower moisture content set, until the total residual deviance was minimized. Extrapolation from lower moisture contents showed that the open symbol at 31 8% moisture content showed greater longevity than would be expected from linear regression (Fig. 4A). This value was then excluded from the data set, since it was clear graphically that 31 8% moisture content is above the uppermoisture-content limit to the negative logarithmic relation between conidia longevity and moisture content. The negative logarithmic relation between σ and moisture content between 5 3 and 21 2% was significant (P 5, r 953). In contrast, that between σ and moisture content between 2 5 and 4 2% was not (P 25). Accordingly, a horizontal line at log σ (s.e. 2) (i.e. σ 44 1 d) is shown in Fig. 4A for observations between 2 5 and 4 2% moisture content, and a negative logarithmic relation for observations between 5 3 and 21 2% moisture content with K (s.e. 247) and C W 3 59 (s.e. 239) for eqn (5). The two lines intersect at 4 6%; i.e. 4 6% moisture content is the lower-moisturecontent limit to the negative logarithmic relation between conidia longevity and moisture content. A previous estimate of C W, derived from the data of Hedgecock et al. (1995), was 2 24 (s.e. 2) (Hong et al., 1997). In fact, the broken line in Fig. 4A shows good agreement between the predictions of longevity provided by the model of Hong et al. (1997) and the independent observations between 8 and 17% moisture content in the current study. The lower estimate of C W from the previous study could have arisen for several reasons. In particular there was a difference in the method of storage. Hedgecock et al. (1995) stored dry conidia of M. fla o iride in a groundnut-kerosene oil mixture, compared to the hermetic air-dry storage in laminated aluminium foil packets used here. Both estimates of C W are low in comparison with those for orthodox seeds of most cultivated crop species, typically in the range (Hong, Linington and Ellis, 1996), but they are close to estimates for seeds of certain tree species (Hong et al., 1996), e.g. C W 3 33 for Liquidambar styraciflua L. (Bonner, 1994).

4 628 Hong et al. Longe ity of Fungal Conidia (9.) A 12.1 (51.6) E 5.3 (12.2) J (76.9) B 1.3 (43.8) F 4.2 (9.9) K (67.8) C 8.6 (34.) G 3.2 (9.3) L Germination (%) (57.9) D 8. (28.2) H 2.7 (9.1) M (16.5) I 2.5 (9.) N Duration of storage (d) 5 1 FIG. 2. Survival curves (% germination s. duration of experimental storage) of conidia of Metarhizium fla o iride stored hermetically at 5 C with 14 different moisture contents from 31 8 (A) to 2 5% (N). Values shown in parentheses after each moisture content are equilibrium relative humidities at 2 C. Note the difference between the three columns in the scales for the period of storage. The fitted curves shown are negative cumulative normal distributions [eqn (2)] where a (s.e. 4) and σ has the values shown in Fig

5 Hong et al. Longe ity of Fungal Conidia A B 12 Germination (%) Duration of storage (d) FIG. 3. Survival of conidia of Metarhizium fla o iride stored hermetically at 13 8% moisture content (fresh weight basis) at 5 C. The curves shown are negative exponentials ( ) or negative cumulative normal distributions ( ) with (A) or without (B) all 14 survival curves constrained to a common origin A B Longevity (σ, d) Moisture content (%, w.b.) Equilibrium relative humidity (%) FIG. 4. Effect of moisture on conidia longevity (symbols show s.d. of the frequency distribution of conidia deaths in time; σ, d) in Metarhizium fla o iride stored hermetically at 5 C. A, The negative logarithmic relation between conidia longevity and moisture content fitted to observations between 5 3 and 21 2% moisture content ( ) is shown by a solid line;, moisture contents below the lower-moisture-content limit or above the upper-moisture-content limit to this relation. B, The negative semi-logarithmic relation between conidia longevity and equilibrium relative humidity fitted to observations between 12 2 and 76 9% RH is shown by a solid line. The broken lines shown are independent of the observations and represent the relations predicted by the viability equations developed previously for Metarhizium fla o iride (Hong et al., 1997). See text for further information. 1 On the other hand, the estimate of the lower-moisturecontent limit to the negative logarithmic relation between conidia longevity and moisture content (4 6%) is in equilibrium with about 1 7% RH at 2 C (Fig. 1). This value is very similar to estimates of 1 12% RH for seeds (Ellis et al., 1988, 1989, 1992). The estimate of the lowermoisture-content limit of 4 6% (or 1 7% RH) has practical significance: it is the value to which conidia should be dried in order to maximize longevity in storage at 5 C, and probably cooler temperatures. A precise estimate of the upper-moisture-content limit to the negative logarithmic relation between conidia longevity and moisture content was not provided by the current results, but it is clearly between 21 2 and 31 8% moisture content. In seeds, the upper-moisture-content limit varies considerably among species, but these values tend to coincide at a seed water potential of about 14 MPa (Roberts and Ellis, 1989), i.e. at seed moisture contents in equilibrium with about 85 9% RH. Since the conidia of M. fla o iride at 31 8% moisture content were in equilibrium with 9 % RH (Fig. 1) and the estimate of σ was only slightly greater than extrapolation from lower moisture contents (Fig. 4A), we suggest that it is likely that the uppermoisture-content limit, in terms of equilibrium relative humidity, may be similar in these fungal spores to those in seeds.

6 63 Hong et al. Longe ity of Fungal Conidia Relations between longe ity and equilibrium relati e humidity The observations between the upper- and lower-moisturecontent limits to the negative logarithmic relation between conidia longevity and moisture content were also analysed in accordance with eqn (6). That analysis showed a significant semi-logarithmic relation between longevity and equilibrium relative humidity between 12 2 and 76 9% RH (P 5, r 941), with the estimates K (s.e. 123) and C R 269 (s.e. 25) (Fig. 4B). The fit of this line was not as good as that provided by eqn (5), but one advantage of this relation is that in orthodox seeds the estimates of the slope (i.e. the value C R ) have been shown to be invariant, at least among 23 species investigated (Ellis et al., 1988, 1989, 199a, b; Roberts and Ellis, 1989). On the other hand, the independent estimates of the values of K E, C R, C H and C Q [eqn (4)] for M. fla o iride predicted conidia longevity in the current study very well (Fig. 4B). The current estimate of C R is broadly similar to that determined previously, 217 (s.e. 21) (Hong et al., 1997) from the results of storage of dry conidia suspended in a groundnut-kerosene oil mixture. These values of C R are somewhat lower than those for orthodox seeds (typically C R is about 35), but in both seeds and conidia the estimates are reasonably close to the generalization that a 1% reduction in equilibrium relative humidity doubles longevity. This current estimate of C R of 269 implies that each 11 2% reduction in equilibrium relative humidity between 1 7 and 8% RH doubles conidia longevity. In conclusion, this study has shown that the response of the longevity of conidia of the entomopathogenic fungus M. fla o iride to moisture content can be quantified, and that the upper- and lower-moisture-content limits to this relation and the sensitivity of longevity to equilibrium relative humidity between these limits are similar to those for orthodox seeds. The research therefore shows that considerable similarities in survival characteristics exist in anhydrous biology among contrasting propagules and that, as shown by the comparison between the broken line and solid symbols in Fig. 4B, conidia survival in air-dry storage is predictable. This augurs well for the reliable maintenance of conidia viability under specified conditions in biological control programmes using entomopathogenic fungi. ACKNOWLEDGEMENTS This research is part of the collaborative Lutte Biologique Contre les Locustes et Sauteriaux (LUBILOSA) programme between the International Institute of Biological Control (IIBC), the Biological Control Programme Centre of the International Institute of Tropical Agriculture (IITA), Cotonou, Benin, and the De partement de Formation en Protection des Ve ge taux (DFPV), Niamey, Niger, funded by the Canadian International Development Agency, the Netherlands Directorate General for International Cooperation, The UK Overseas Development Administration, and the Swiss Development Cooperation. We thank Ms Jane Gunn for technical assistance. LITERATURE CITED Baker RJ, Nelder JA The GLIM system, release 3. Oxford: Numerical Algorithms Group. Bonner FT Predicting seed longevity for four forest tree species with orthodox seeds. 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