Water relations of fungal spore germination - Springer Link

9 downloads 0 Views 570KB Size Report
Summary. A simple mathematical model, based on the physiology of spore germination of Penicil- lium roqueforti and Trichoderma viride TS, is pro- posed and ...
App//ed

Appl Microbiol Biotechnol (1988) 29:586-592

Microb/o/ogy Biotechnology © Springer-Verlag 1988

Water relations of fungal spore germination Patrick Gervais, Jean-Philippe Fasquel, and Paul Molin Laboratoire de Biologie Physico-Chimique, ENS.BANA, Campus Universitaire, F-21000 Dijon, France

Summary. A simple mathematical model, based on the physiology of spore germination of Penicillium roqueforti and Trichoderma viride TS, is proposed and tested to determine germination kinetics of filamentous fungi. The influence of water and of the nature of the solute used to depress the water activity on conidial germination of these two fungi are discussed. The water activity value of the medium is the main factor but the water molar fraction seems to explain certain observed variations in germination kinetics. The best solutes for germination are those which present the greatest deviation from Raoult's law.

Introduction Germination of a mold spore is a physiological reaction of a resting cell to modifications of the environmental conditions. The resting state of a spore is controlled by the internal presence of metabolic blocks, nutrient penetration barriers, selfinhibitors, and low water content (Smith 1978). After a certain time lag, three successive structural changes are observed during germination (Gottlieb 1978): the swelling of the mold spore, the emergence of a germ tube, and the elongation. Water is a predominant part of germination, and hydration of the medium is an important parameter (Snow 1949). Germination has been observed in pure water for Alternaria and Cladosporium (Dickinson and Bottomley 1980). Penicillium frequentans and Trichoderma viride have been identified as strains with self-feeding spores (Sheridan and Sheeman 1980). However, other authors have shown that the conidia of P. notatum and T. lignorurn need carbon and nitrogen to gerOffprint requests to: P. Gervais

minate (Martin and Nicolas 1970). When the availability of water is limited, the water activity (aw) of the medium strongly influences the mold development (Gervais et al. 1988a). This parameter is widely used for quantifying availability and energy state of water. Nevertheless, aw alone cannot explain the biological variations; when the medium aw is adjusted by solutes the nature of the solute has an effect (Beuchat 1983). Only few authors have tried to describe germination of conidia as a function of time. An empirical model for spore germination of Alternaria solani was described, and further extended in order to develop a complex box model of germination (Waggoner and Parlange 1975). A model proposed for conidial germination in Colletotrichum graminicola (Lapp and Skoropad 1976) fitted the data well, but the parameters had little biological significance. The model proposed in this work is based on the observation of germination kinetics and the parameters involved take the physiology of germination into account.

Materials and methods Theoretical analysis of germination Germination of a mold spore is considered as a classical first order system where the basis hypothesis is the proportionality between the rate of germination and the corresponding number of ungerminated conidia: At time t = 0 all the spores (No) were ungerminated and after time lag to, conidia began to germinate. At time t, N spores were germinated and the basic hypothesis allows to write: dN d (t - to)

k- (No- N)

k = constant of velocity.

(1)

587

P. Gervais et al.: Water relations of fungal spore germination After integration (No - N) = e--k( . . . . )" a

(2)

a = constant of integration. At t = to, N = 0 and then a = No N =l-e-k< No

.... )

(3)

With k = l / t , and introducing a maximum ratio of germination K, the final equation was: N --= No

K-(1-e

- ( . . . . )/4)

(4)

N is the ratio of germinated spores at t No The model was described by three parameters: K = maximum ratio of germination to = time lag )~=time constant, corresponding to the time needed to reach 63% of the maximum germination (K). Then I/A was an estimation of the rate of germination.

ol-agar mixture. The conidia development is limited on this medium as compared to germination on a nutritive medium. Media of aw values 0.950 and 0.850 for the solute influence study were prepared by mixing glycerol, sorbitol, maltose, sucrose, fructose or propylene glycol with water and agar (1.5% w/w). The medium without these substances had a aw value of 0.999. The effective solute concentrations were calculated using the Norrish's equation (1966): a w = ( 1 - X ) e -K'x2

(5)

aw: water activity value X: solute molar fraction K: depending on the solute (Chirife et al. 1980) The media were sterilized by autoclaving at 120°C for 20 min. Water activity values were checked before and after cultivation: For 0.95 and below with a Sina-Scope instrument (Sina, Zurich, Switzerland). For 0.96 and above by refractometry or microosmometry (microosmometre Roebling, D-1000 Berlin). Calculated values, which never varied by more than 0.005 aw units from those measured, have been used.

Cultivation Fungi Trichoderma viride TS, which is a mutant with reduced sporogenesis obtained by Dr. T. Staron (INRA, Luc6, France) from T. viride recommended for protein production from vegetable by-products, and Penicillium roqueforti, strain n ° 18 from the Laboratory of Biotechnology (ENS.BANA, Dijon, France), used in cheese production, were studied.

Spore production In order to study the influence of medium hydration on germination, conidia were produced on the modified Morton medium (Morton 1960), diluted to 1/4 strength and solidified with 15 g of agar per liter. The medium was buffered to pH 5.0, and the water activity value was 0.970 to produce conidia from P. roqueforti, and 0.980 from T. viride TS as shown previously (Gervais et al. 1988b). Potato Dextrose Agar for P. roqueforti, and Emerson's Yp Ss agar (agar 18 g, soluble starch 15 g, yeast extract 4 g, K2H PO4 1 g, MgSO4- 7 H20 0.5 g, HzO 1 1) for T. viride TS were used to obtain spores to study solute influence. After a 10 days growing period at 25°C for P. roqueforti and at 30°C for T. v&ide TS the conidia were recovered by moderate shaking of the colony with a mixture of sterile distilled water and Tween 80 (1%0 v/v). The final concentration of the conidia in the suspension was about 10 7 - 10 8 conidia/ml. Conidia were stored at 4 ° C.

Medium was poured as thin layer (5 ml medium/dish) into a Petri dish (80 mm diameter). Cultures were inoculated by rubbing approximately 0.01 ml of the spore suspension gently on the medium until complete absorption of inoculum water by the medium. Three Petri dishes of a fixed aw value were stored into a translucent polyethylene food storage box containing 200 ml of an appropriate water-glycerol solution to control the relative humidity of the atmosphere.

Examination Each germination kinetic was observed with a microscope on the same marked point where spores were well isolated, to avoid problems caused by variations in time lag. Petri dishes were examined by transmitted light microscopy (100 x ). Three repetitions were made for each condition in the hydration influence study and each measurement was the mean of two countings (100 spores/counting). The criterion used for germination was the observation of a germ tube of length at least to 1.5 times the diameter of the spore examined (Martin and Nicolas 1970).

Evaluation of the model A derivative-free non-linear regression analysis of equation (4) for each time curve of germination was performed using the PAR software from BMDP (Statistical Software 1983, UCLA, Calif., USA).

Media

Results

To study the influence of hydration, glycerol was used as the water activity depressor because of its well known compatible solute properties (Hocking and Pitt 1979). Preliminary measurements have shown that spores of the studied strains need oxygen to germinate, and that this could occur on a water-glycer-

Application o f the m o d e l

and discussion

One of the three repetitions observed for each f u n g u s a n d f o r t h e s a m e aw is s h o w n f o r aw -- 0.99,

588

P. Gervais et al.: Water relations of fungal spore germination

due to the variations in the time lag between repetitions. The application of the model was validated as follows: for each water activity value, each fungus, and each value, the fit of the regression line was calculated with a Chi-Square Test and was found to be highly significant (1% level). Thus, equation (4) may interpret the germination data using the three parameters K, to, and 2, of a real biological significance.

loo

A 50,

o

Influence o f water on conidial germination

0 $

I

21o

3O

T£me

(hours)

In order to study the influence of the water activity of the m e d i u m on germination, the m e a n values of the parameters K, to, and 2' for each aw are presented in Table 1 for P. roqueforti and T. viride TS. The m i n i m u m aw values for germination observed were 0.90 for T. viride TS and 0.86 for P. roqueforti. C o m p l e t e germination of P. roqueforti (100%) was observed for a~ values ranging from 0.98 to c.a 1 and T. viride TS at 0.98 to 0.99 aw.

1 00 J

..,M r. o o

o

Table 1. Mean values of the equation (4) parameters (K, to,

o

and 2) for the germination of P. roqueforti :A, and of T. viride TS :B; Confidence intervals of the mean values for a confidence level equal to 0.90 are presented in brackets A

o~

,

,

10

i

2o

30

40

TL,.e

(hours)

Parameter water activity

K

e_

o

4J

0.99 0.98 0.97 0.96 0.95 0.92 0.90 0.88

C g

@ o

I

J

[_

(h) (1.7) (0.7) (o.o) (5.0) (6.8) (1.9) (3.6) (18.8) (15.9)

99.4 99.6 100.0 96.2 96.4 92.2 82.7 88.0 35.2

2 (h)

to

(% 58.6 14.4 8.2 10.6 13.1 12.9 28.4 62.1 157.6

(68.0) (2.6) (3.5) (1.0) (0.5) (0.6) (4.0) (72.6) (10.9)

(53.1) (1.3) (1.4) (0.9) (3.1) (2.9) (9.3) (91.7) (188.6)

44.7 3.6 6.1 4.7 7.1 8.2 21.6 76.2 147.7

B

i

Time

(hours)

Fig. 1. Germination kinetics of P. roqueforti ( 0 ) and T. viride TS (©) at aw=0.99 :A, P. roqueforti ( I ) and 7". viride TS ([~) at aw=0.95 :B, P. roqueforti ( 4 ) and T. viride TS (A) at aw = 0.92 :C

aw-0.95, and aw=0.92 in Fig. 1. Both the observed data and the theoretical line produced by the regression of the data using equation (4) are presented. The mean of the three values used for the same aw cannot be used to validate the model

Parameter water activity ~-1 0.99 0.98 0.97 0.96 0.95 0.94 0.92 0.90

K

85.6 98.6 100.0 74.6 67.6 58.9 43.8 44.6 20.7

(14.9) (4.2) (0.0) (22.2) (14.7) (4.8) (4.8) (26.6) (7.4)

2

to (h)

(%) 11.3 5.3 9.9 13.1 12.7 16.5 16.2 46.8 21.6

(h) (3.2) (9.0) (2.7) (1.1) (1.7) (1.8) (0.8) (48.7) (12.5)

21.3 28.4 23.5 11.9 15.9 12.8 19.1 ~48.3 313.7

(12.3) (13.2) (7.9) (4.8) (7.1) (9.4) (4.3) (272.0) (340.6)

P. Gervais et al.: Water relations of fungal spore germination

For lower aw values, percentages of maximum germination decreased regularly. The shortest time for germination occured between 0.94 and c.a 1 for T. viride TS, with a minimum at 0.99, and for P. roqueforti between 0.95 and 0.99, with a minimum at 0.98. The same limits of water activity were observed for the highest rates of germination, evaluated by 1/1. Maxima were then at 0.97 for T. viride TS and at 0.99 for P. roqueforti. P. roqueforti germinated more rapidly and more completely than T. viride TS at all aw values studied. The water activity value of the medium close to 1 was not a good hydration condition for the germination of both fungi. Important variations in the time lags and in the rapidity of germination were observed. The high mean values at the low water activities (below 0.92), indicated difficulties by the conidia to germinate when the environmental conditions became unfavourable. In general, water activity values from 0.94 to 0.99 were favourable to the germination of P. roqueforti, and the most favourable values for T. viride TS were 0.94 to c.a 1. The use of the three parameters K, to, and 1 for the model was justified by the study of the correlation coefficients: no significant relationship was observed between these parameters. The minimum aw value of 0.86 which allowed the germination of P. roqueforti was higher than 0.83 found previously (Magnan and Lacey 1984) with glycerol agar medium. But the absence of nutrients reduces the range of aw values where germination and growth are possible (Snow 1949). The influence of the low water activity values on germination is not well understood (Charlang and Horowitz 1974). The loss of an essential substance for germination from Neurospora crassa, Aspergillus nidulans and P. chrysogenum at low water activity has been suggested (Charlang and Horowitz 1971; Charlang and Horowitz 1974). According to Gottlieb (1978) water allows the diffusion of self inhibitors out of the spore. In all cases, the membrane permeability is important. Dehydration of the membrane would produce a molecular rearrangement of phospholipids leading to a rigidification and a loss of permeability of the membrane (Quinn 1985). The rearrangement would occur at an aw value which corresponds to the minimum water activity value observed for germination. If germination is allowed by the diffusion of self inhibitors out of the spore, the concentration

589

100

M

5O

o

i

J

o

Wo-W

i

It-

[ 25

(g/lOOg wet medium)

Fig. 2. Influence of water content on K parameter of equation (8); P. roqueforti (0), T. viride TS ( ~ )

of inhibitors in the conidia would be inversely proportional to the water content of the medium, until the equilibrium water content (W0) or water activity (awo) is reached. This value which corresponds to the maximum germination ratio of the fungus is equal to 0.98 for both fungi as shown in Table 1. So below awo, the K parameter of the germination (see Eq. (4)) would be related to the water content of the medium by the following equation: K = A. W

(6)

With A-- constant of proportionality and W = water content If K0 is the maximal value of the parameter K corresponding to a water content of Wo Ko = A. Wo

(7)

From (6) and (7) K = - A ( W o - W ) + Ko

(8)

Equation (8) was compared to the experimental data in Fig. 2. The correlation coefficients of the corresponding regression were significant (5% level). This type of model would be interesting but must be confirmed.

Influence of the solute on spore 9ermination Kinetics of germination of the two fungi at aw=0.95 are presented in Fig. 3 for the solutes

590

P. Gervais et al.: Water relations of fungal spore germination

A 0

"~

50.

e

g~J o ~4

~o

Time

(hours)

Time

(hours )

B

30.

2O.

10-

16

18

20

22

Fig. 3. Germination of P. roqueforti :A and T. viride TS :B at aw=0.95 in glycerol (Ill), sorbitol (O), maltose (V), sucrose (A) and fructose V)

studied. The previous model was not tested on these curves because of the short duration of the experiment (28 h). As shown previously, germination of P. roqueforti was more complete than T. viride for whatever solute. Germination of the two fungi was more complete in sugar solutions than in polyol solutions. In fructose and maltose the germination intensity was 2 times greater than in glycerol which was the most favourable polyol. Propylene glycol was also tested in a previous experiment (Feodoroff 1986); P. roqueforti only showed a very limited germination and T. viride could not germinate. The same experiment at aw=0.85 revealed greater differences between solutes, but the results could not be quantified: P. roqueforti germinated in all solutes except glycerol and propylene glycol when T. viride did not germinate in any solution. If the specific solute effect is not evident for high water activities for growth of microorgan-

isms as observed by numerous authors (Scott 1957; Christian 1955), this specific effect has been pointed out for low aw (Christian 1980). Specific solute effects on the rate of bacterial spore germination have already been described (Jakobsen 1985), but only minor effects were observed on the time lag of mold germination (Hocking and Pitt 1979). Thus, low a,,¢ levels used in this experiment are certainly related to the strong specific solute effect observed. For propylene glycol and polyethylene glycol (Vaamonde et al. 1982) such an effect could be attributed to inhibiting effects of these molecules on metabolic activities of fungi. For other solutes which are all compatible solutes, the interpretation is more difficult. Life adaptation to low water activities is explained by the interaction of such solutes as polyols or sugars (Brown 1978) and amino acids (Measures 1975) with molecules of biopolymers (enzymes, DNA, membrane phospholipids). These compounds are assumed to enhance the stabilization and the maintenance of functional properties by hydrophobic interactions (Brown 1978) or by supplying hydrogen bonds in the absence of water (Clegg et al. 1982; Crowe and Crowe 1982). In this case, it would be understandable why solutions do not only act towards microorganisms through their colligative properties and water activity can not be related to such effects. There is no valid theory at present, to predict the influence of the solute on the metabolic activity of microorganisms, nevertheless, some information could be extracted from this work. A solution is said to be ideal when molecular interactions in the solution are the same as in pure compounds. In this case, application of Raoult's law leads to aw=Xw with Xw: molar fraction of water in the solution. Thus, an ideal and compatible solute will act only through its colligative properties for a physiological activity. This deduction is also true for a very dilute solution which follows Henry's law. So, the stronger the molecular interaction the greater the deviation from these two laws. Such a deviation can be estimated by the comparison between Xw and aw values of a solution, proposed in Table 2 for the five Table 2. Water molar fraction of the media at aw values 0.95 and 0.85

Glycerol

Sorbitol

Maltose

Sucrose

Fructose

aw 0.95

0.953

0.954

0.958

0.960

0.961

a~, 0.85

0.868

0.873

0.895

0.904

0.905

P. Gervais et al. : Water relations of fungal spore germination

591

solutes used. For a real solution this difference can be expressed by the water coefficient of activity 7rw in the solution: aw = ywXw (with ~'w= 1 for an ideal solution). This difference, which can be related to the molecular weight of the solute and can be attributed to the ability of the molecule to interact with water, increases from glycerol to fructose. Comparing these results to Fig. 3 shows that the sugars, which present a greater deviation from the Raoult's law than the polyols, are favouring the kinetics of germination of the fungi. Nevertheless this observation must be nuanced for solutes which present quasi-similar intensities of deviation from the ideal behaviour as glycerol and sorbitol or maltose and sucrose. For both couples of solutes the germination curves are inverted considering the ideal behaviour. These phenomena could be explained by specifical affinities between the solute and biological macromolecules. The ability of glycerol to permeate the cell walls, since sorbitol do not permeate, can interfere in this area. Meanwhile such an analysis can be used to predict the capacity of a solute to protect a microorganism against a decrease of water content in the medium. The time lag of germination which is comparable for all solutes seems to be essentially a function of colligative properties of the solutions but the kinetics of germination appear to be strongly dependent on the type of solute. The following explanation can be proposed: more a solute is deviating from ideal behaviour and more its molecular conformation can induce numerous hydrophilic interactions. Thus this solute can easily interact with the polar groups of the membrane corresponding to phospholipids and enzymes hydrophilic parts. When associated to drastic conditions as temperature or hydration stress, such a stabilization would allow to maintain the fonctionnality of these biopolymers essentially in order to assume the membrane permeability. Previous conclusions (Anchordoguy et al. 1987) that trehalose is a very efficient solute interacting with the membrane structure are in complete agreement with these results.

Brown AD (1978) Compatible solutes and extreme water stress in eukaryotic microorganisms. Adv Microb Phys 17:181242 Charlang GW, Horowitz NH (1971) Germination and growth of Neurospora at low water activities. Proc Nat Acad Sci 68: 260-262 Charlang GW, Horowitz NH (1974) Membrane permeability and the loss of germination factor from Neurospora crassa at low water activity. J Bact 117:261-264 Chirife J, Ferro-Fontan C, Benmergui EA (1980) The prediction of water activity in aqueous solutions in connection with intermediate moisture foods IV. J Food Technol 15:59-70 Christian JHB (1955) The water relations of growth and respiration of Salmonella oranienbur# at 30 oC. Aust J Biol Sci 8: 490-497 Christian JHB (1980) Specific solute effects on microbial water relations. In: Rockland LB, Stewart GF (eds) Water Activity: Influence on Food Quality. Academic Press, London, pp 825-854 Clegg JS, Seitz P, Seitz W, Hazlewood CF (1982) Cellular response to extreme water loss: the water replacement hypothesis. Cryobiology 19:306-316 Crowe JH, Crowe LM (1982) Induction of anhydrobiosis: membrane changes during drying. Cyrobiology 19:317328 Dickinson S, Bottomley R (1980) Germination and growth of Alternaria and Cladosporium in relation to their activity in the phyllophane. Trans Br Mycol Soc 74:309-319 Feodoroff T (1986) Influence des parametres d'hydratation du milieu sur la germination de champignons filamenteux. D.E.A. Sciences des Aliments, Universit~ de Bourgogne, Dijon, France Gervais P, Bensoussan M, Grajek W (1988a) Water activity and water content: comparative effects on the growth of Penicillium roqueforti on solid substrate. Appl Microbiol Biotechnol 27: 389-392 Gervais P, Grajek W, Bensoussan M (1988b) Influence of the water activity of a solid substrate on the growth rate and sporogenesis of filamentous fungi. Biotechn Bioeng 31:457-463 Gottlieb D (1978) The Germination of Fungus Spores. Meadowfield Press, Durham Hocking AD, Pitt JI (1979) Water relations of some Penicillium species at 25 oC. Trans Br Mycol Soc 732:141-145 Jakobsen M (1985) Effect of Aw on growth and survival of bacillaceae. In: Simatos D, Multon JL (eds) Properties of Water in Foods. Martinus Nijhoff, Dordrecht, pp 259272 Lapp MS, Skoropad WP (1976) A mathematical model of conidial germination and appressorial formation for Colletotrichum #raminicola. Can J Bot 54:2239-2242 Magnan N, Lacey J (1984) Effects of temperature and pH on water relations of field and storage fungi. Trans Br Mycol Soc 82:71-81 Martin JF, Nicolas G (1970) Physiology of spore germination in Penicillium notatum and Trichoderma lignorum. Trans Br Mycol Soc 55:141-148. Measures JC (1975) Role of amino acids in osmoregulation of non-halophilic bacteria. Nature 257:398-400 Morton AG (1960) The induction of sporulation in mould fungi. Proc Royal Soc London 153:548-569 Norrish RS (1966) An equation for activity coefficients and equilibrium relative humidity of water in confectionary syrups. J Food Technol 1:25-39

References Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH (1987) Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology 24:324-331 Beuchat LR (1983) Influence of water activity on growth, metabolic activities and survival of yeasts and moulds. J Food Prot 46:135-141

592

P. Gervais et al.: Water relations of fungal spore germination

Quinn PY (1985) A lipid phase separation model of low-temperature damage to biological membranes. Cryobiology 22:128-146 Scott WJ (1957) Water relations of food spoilage microorganisms. Adv Food Res 7:83-125 Sheridan JJ, Sheeman PJ (1980) Development of a technique for the germination of fungal spores. Irish J Agricult Res 19:155-159 Smith JE (1978) Asexual sporulation in filamentous fungi. In: Smith-Berry (ed) The Filamentous Fungi, vol 3" Developmental mycology. Edward Arrold Publishers, pp 214-235 Snow D (1949) The germination of mould spores at controlled humidities. Ann Appl Biol 36:1-13

Vaamonde G, Chirife J, Scorza OC (1982) An examination of the minimal water activity for Staphylococcus aureus ATCC 6538 P growth in laboratory-media adjusted with less conventional solutes. J Food Sci 47:1259-1262 Waggoner PE, Parlange JY (1975) Slowing of spore germination with changes between moderately warm and cold temperatures. Phytopathology 65: 551-553

Received May 9, 1988/Accepted August 17, 1988