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The rôle of saprophytic fungi in antagonism against Drechslera sorokiniana (Helminthosporium sativum) on agar plates and on rye leaves with pollen
Physiological
Plant Pathology
(1973)
3, 195-205
The rdle of saprophytic fungi in antagonism against Drechslera sorokiniana (Helminthosporium on agar plates and on rye leaves with pollen
sativum)
N. J. FOKKEMA Phytopathologisch (Accepted
Laboratorium
for publication
“Willie
September
Commelin
&h&m”,
Baarn,
The Netherlands
1972)
About 50 isolates from each of the four dominant groups in the phyllosphere of rye (Se& cereale) were tested for inhibitory effects against Drechslera sorokiniana (Helminthosporium sativum) in agar culture. The most antagonistic fimgus was Aureobasidium pullulanr, followed by the Sporobolomyces spp. group and then the “White yeasts” (mainly CryPfococcur spp.) . There was considerable variation in inhibitory activity within each group on agar. Isolates of Cladosporium spp. did not show any antagonistic effect against Drechslera in vitro. Inoculations of living rye leaves with D. sorokiniana and isolates from each group of saprophytic fungi always resulted in a remarkable reduction of infection in the presence of rye pollen, contrasting with the varying degrees of antagonistic effect observed in agar culture. Development of superficial mycelium of the pathogen on the rye leaf surface and the subsequent severity of necrosis are positively correlated. Both on leaves and on slides coated with agar, it was the enhanced superficial mycelial growth of the pathogen in the presence of pollen which was inhibited by the saprophytes. Germination of the Drechslera spores was not affected.
INTRODUCTION
Antagonistic action by saprophytic fungi against plant pathogens may be based on competition for nutrients, on production of antibiotics or on induction of phytoalexins. The relevant literature was recently reviewed by Van den Heuvel [IL’]. Antagonism of fungi in relation to Helminthosporium diseases of cereal leaves has been studied by Porter [18], Asare-Nyako [Z] and Akai & Kuramoto [I]. Most of the research gave more attention to the inhibition of the pathogen’s development on agar by a great variety of micro-organisms, than to the effect of the natural microflora on the infection. However, Akai & Kuramoto demonstrated that Candida, a yeast dominating on rice leaves, could reduce infection by Cochliobolus miyabeanus by about 50%. Cladosporium, a dominating hyphal fungus in the phyllosphere, appeared to reduce infection of tomatoes and strawberries by Botrytis cinerea [3, 171. Recent studies have revealed that pollen deposited on fruits or leaves of anemophilous plants can stimulate fungal infection [S, IO, II, 20, 24. The objective of the present study was to examine whether this “pollen effect” was reduced by antagonistic activities of the saprophytic mycoflora under field conditions. Previous studies [II] had shown that field inoculations of rye leaves with Helminthosporium sativum just after flowering resulted in a marked increase of infection compared to the infection which occurred after inoculations before flowering. When rye pollen
196
N. J. Fokkema
was added to leaves already bearing a natural pollen deposit, stimulation of infection did not occur. When pollen was added to comparable leaves upon which pollen deposit had been prevented, infection was stimulated. These results were explained [II] by the hypothesis that the natural pollen deposit stimulated an antagonistic population of saprophytes, which reduced infection by H. sativum in the presence of pollen. The saprophytic mycoflora of rye leaves can be separated into four dominant groups: Cladosporium spp., Aureobasidium pullulans (De Bary) Arnaud, the pink yeast Sporobolomyces roseusKluyver & v. Niel and “White yeasts”, mainly C~ptococcus spp. About 50 isolates of each group were tested for antagonistic properties in dual cultures with Drechslera sorokinianat (Sacc.) Subram & Jain (syn.: Helminthosporium sativum Pamm., King & Bakke). Several of the saprophytic isolates were selected, and their effects on infection of leaves by D. sorokiniana were studied. Attempts were made to correlate their antagonistic properties on the leaf surface with those observed in dual cultures. It has previously been suggested that such a correlation may not exist [I, 3, 121. MATERIALS AND METHODS Antagonism on agar plates
The saprophytes were obtained by shaking rye leaves in water, from which isolations of fungi were subsequently made [II]. They were propagated on PDA slants at 23 “C in darkness. The strain of D. sorokiniana, the conidial state of Cochliobolus sativum (Ito & Kuribayashi) Drechsler ex Dastur, was cultured on oatmeal agar slants at 23 “C in darkness.
FIG. 1. Diagram of the mode of inoculation of agar plates with D. sorokiniana and like test organism. Parameters for inhibition are the width of the zone of inhibition the percentage inhibition of radial growth [ 100 x (rl - rs) /rJ.
a yeast(d) and
After culturing for about 2 weeks, a streak (4 cm) of the yeasts or the yeast-like fungus A. pullulans was made on PDA plates (10 ml per Petri dish of 9 cm diameter) as shown in Fig. 1. In the case of the Cladosporium isolates a loopful of the spore suspension was placed 2 cm from the margin of these plate. Opposite the saprophytes, at a distance of at least 3 cm, a loopful of a spore suspension of D. sorokiniana, containing about 20 conidia, was deposited. After incubation for 6 days at 23 “C t Sensu Ellis
[9].
R6le
of fungi
against
D. sorokiniana
197
in darkness, inhibition of the pathogen’s development was assessed by two parameters, viz. the percentage of inhibition of radial growth [lo0 x (rr-rs)/rJ and the width of the zone of inhibition measured at the smallest distance between both colonies (d), as illustrated in Fig, 1. The use of two parameters allows discrimination between inhibition of the growth of the pathogen and the type of interaction, such as inhibiMoreover, the various growth rates of the saprotion at a distance or by contact. phytes will affect the radial growth of the pathogen independent of their antagonistic action, whereas the zone of inhibition remains unaffected. On the other hand, the width of the zone of inhibition may be affected also by retardation of the growth of the saprophyte. Antagonism
on rye leaves
Summer rye, Secale cereale L. cv. “Petkuser”, was used as the host and grown as described earlier [II]. The penultimate leaves were inoculated with a suspension consisting of I.5 to 3.5 x lo5 spores of D. sorokiniana, 3 x 10’ spores (cells) of an isolate of the yeasts or A. pullulans or l-25 x 10’ spores of a Cladosporium isolate, and 30 mg of rye pollen per 1 ml of O*1o/o Tween 80. As a control, leaves were inoculated with D. sorokiniana alone, with or without pollen addition. Generally, a set of eight leaves received the same treatment. The fungi were cultured similarly as for the in vitro studies. Yeasts and Aureobasidium, however, were grown for 6 days instead of 2 weeks. Spores and yeast cells were suspended in O-1o/0 Tween 80 by scraping the cultures with an inoculation needle followed by shaking. Masses of agar and large mycelial fragments were removed by filtering through glasswool. Before inoculation, the leaves were drawn between the moistened thumb and index finger to disturb the wax layer ensuring a uniform distribution of the inoculum drop. Then 0.1 ml of the inoculation suspension was pipetted on the leaf base and spread over the whole surface by drawing the leaf between thumb and index finger. Immediately after inoculation the plants were placed in large transparent plastic covered cages lined with moist filter paper. The temperatures varied between 18 and 25 “C and relative humidity from 85 to 95%. The areas of necrosis were assessed 7 days after inoculation by means of a planimeter and expressed as a percentage of the leaf surface area. In some experiments, the pre-infection development of the pathogen was assessed 3 days after inoculation. Therefore additional sets of eight leaves were inoculated. On these leaves spore germination and the development of superficial mycelium on leaf parts, 6 to 9 cm from the base of the lamina, were studied on transparent adhesive tape replicas [II.] The superficial mycelial development was assessed by projecting photomicrographs from the replica on to a paper. Photomicrographs were made from three distinct areas on each replica (total area of about 0.8 mm2 per replica). The length of the hyphae was measured with a curvimeter and expressed in units of 25 pm/mm2 of leaf surface. The remaining parts of the leaves, used for the replicas, were used for the assessment of the development of the added saprophytes by plating out the washing water [II]. The capacity of the saprophytes to neutralize the pollen effect was expressed as the relative inhibitory effect (R.I.E.), which may be calculated by 100 x (DP DPS)/(DPD), where DP, DPS and D are the results after inoculation with Drechslera plus pollen (DP), with DP plus a saprophyte (DPS) and with Drechslera
N. J. Fokkema
198
alone (D). Either the effect of the saprophytes on the superficial mycelial development of the pathogen or on the necrosis was expressed as the R.I.E. The data from each of the experiments were subjected to the Wilcoxon’s two sample test. RESULTS Antagonism
on agar plates The behaviour of the 48 Cladosporium isolates, mostly belonging to C. herbarum (Pers.) Link ex Fr., against D. sorokiniana, was very uniform. Both colonies grew undisturbed until they came in contact with each other. Their mycelia did not intermingle. 1
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FIG. 2. Antagonistic properties of isolates of Aureobmidium (a), Sporobolomyces (b) and “White yeasts” (c) against D. s~okiniana in dual cultures, expressed as the width of the zone of inhibition and the percentage inhibition of radial growth of D. sorokiniana. + indicates the isolates used in in viva experiments.
R61e of fungi
against
199
D. sorokiniana
The behaviour of the yeasts and of the yeast-like fungus A. pullu&s was much more variable. These experiments were repeated three times. The largest time interval between replicates was at least 4 months, which may give an indication of the stability of the antagonistic properties of a particular isolate over this period. The mean results for each isolate are given in Fig. 2. Nearly all isolates of the A. pullulans group caused inhibition at a distance. The inhibition of the radial growth, as compared with the zone of inhibition, was more pronounced than in the yeasts, but this was mainly due to the fact that their outgrowth was much less than that of A. pullulans. Unlike the yeasts, the development of some isolates of A. pullulans towards the Drechslera colony was inhibited too, indicating mutual inhibition. About 70% of the S’orobolomyces isolates caused inhibition at a distance, others could be approached by Drechslera until contact was made, or were even overgrown by it, resulting in a negative value for the distance between the colonies. Only 30% of “White yeasts” caused inhibition at a distance, the remaining isolates were overgrown. Forty-four per cent of the isolates inhibited the radial growth by 20% or less. The range between the three values for d and 100 x (rl-rs)/r]. of the individual isolates in the three experiments gives an impression of the reproducibility of the antagonistic properties in vitro. The mean value of these ranges concerning all isolates of A. pullulans amounted to 1.5 mm and to 8*Oo/o for the distance and for the percentage inhibition of radial growth respectively. Concerning the isolates of Sporobolomyces, these figures were 4.3 mm and 13*5%, while for the “White yeasts” the means of the ranges were 3.9 mm and 16.3%. Considering these values and the scales used for both parameters in Fig. 2, it may be concluded that the reproducibility of the antagonistic properties in vitro of a single isolate is slight. Antagonism
on leaves
The isolates of C. herbarum did not show any antagonistic effect to D. sorokiniana in vitro. Two isolates were taken to investigate the effect of C. hzrbarum on the infection of rye leaves by D. sorokiniana, in the presence of pollen (Table 1). The infection, TABLE Effect of adding C. herbarum
Treatment
1
isolates to the inoculum on the stimulatoly of rye leaves by D. sorokiniana
Relative inhibitory
Mean necrotic leaf area (%) Exp. 1 Exp. 2 Exp. 3
D. sorokiniana (D) D + Pollen (DP) DP + Clad. 42 DP f Clad. 33
3** 57 10** 36*
* P< 0.95, ** P< O*Ol, the DP treatment.
levels
1** 72 36* 42** of significance
Exp.
6** 64 17** 6** for
the
effect of pollen on infection
1
87 39 differences
Exp.
2
50 42 with
the results
effect Exp.
3
82 100 after
which is normally low, was highly stimulated when pollen was added. Addition of Cladosporium spores reduced the pollen effect to a considerable degree. Both isolates behaved similarly, and a third C. herbarum isolate, used in a single experiment, gave the same results. Three days after inoculation, the addition of Cladosporium had
200
N. J. Fokkema
resulted in a colonization varying from 24 000 to 67 000 propagules of Cladosporium per cm2 of leaf surface, as assessed by plating out the washing water. The superficial growth of mycelium of D. sorokiniana could not be measured because it was impossible to distinguish it from mycelium of C. herbarum. For comparison of the antagonistic effects of the yeast-like fungi in vitro and in vivo, three isolates of each group were selected for in vivo studies. These isolates, already indicated in Fig. 2, represented the most inhibitory, the moderate or the least inhibitory isolates (Table 2). The experiments were set up in the same way as those with Cladosporium. In most experiments, the superficial mycelial development of Antagonistic
properties
against
D. sorokiniana Zone d (mm)
Isolatesa Aureobasidium
No. No. No.
7 39 49
1 3 5.3
c~ptococcus
No. No. No.
43 50 36
-9-5 -0.7 1.5
Sporobolomyces
No. No. No.
21 30 13
-2
TABLE 2 on agar of yeast-like
of inhibition Range (mm)
3.3 443
0 8 1 2 2.5 6 5 1.5
saprophytic
fungal
isolates
Inhibition on radial growth (%) 100x (rl-r2)/rl Range (%) 52 50 54
13 10 6
8 20 30
7 10 2
21 35 37
17 6 17
a A.7, A.39, A.49 were identified as -4. pullulans (De Bary) Arnaud; (Kuff.) Skinner var. laurentii; C.50 as C. laurentii var. javescens Lodder albidw (Saito) Skinner var. d#lums (Zach) Phaff et Fell; S.21, S.30, as S. roseus Kluyver et V. Niel.
C.43 as C. laurentii et V. Rij; C.36 as C. S.13 were identified
was also determined. The results are presented as values of the relative effect (Table 3). On average the isolates of Aureobasidium, Cryptococcus and S’orobolomyces reduced the stimulation of the infection induced by pollen by a half. The mycelial development of D. sorokiniana was affected to a comparable degree. Contrary to the effects in vitro, no significant difference was found between the general antagonistic action of the three groups, nor in the antagonistic action of the selected isolates within a group. Three days after inoculation, the addition of yeast-like micro-organisms resulted in colonization varying from 30 000 to 600 000 cells of these micro-organisms per cm2 of leaf surface. Within these limits no effect of the concentration of the antagonist on its action could be noticed. In general the addition resulted in about 180 000 cells per cm2. Such a density can be reached under favourable conditions after flowering in the field before yellowing [II]. Th e colonization of the leaves to which suspensions of micro-organisms had not been added seldom reached 1 o/0of the colonization of leaves inoculated with saprophytes. D. sorokiniana
inhibitory
Analysis
of
the reduction of the pollen efect
The results of Table 3 strongly suggest that the reduction of infection was caused by a corresponding reduction of the mycelial growth over the leaf surface. Such a reduction might diminish the number of penetration sites, and consequently reduce
Rale
of fungi
against
201
Lhorokiniana TABLE
3
Effect on subsequent infection of adding yeast-like components of the phyllosphere Jora to pollen and D. sorokiniana on rye leaves. Reduction of the ‘tpollen effect” is expressed as the relative inhibitory effect (R.I.E.), calculated for the mean results of several exjeriments Addition of micro-organisms? Aureobasidium
cryptococcus
Sporobolomyces
mycelial
R.I.E. on development
No. 7 No. 39 No. 49 Mean
6,72* loo,* 85** 55,117**
No. 43 No.50 No.36 Mean
45,49 lo&** 71,**
No. 21 No. 30
66,70 41, 67,*
No. 13 Mean
91,*
39 92 86 72 47 96 58 67
89** 46
90,t
R.I.E. Mean
89,*
92,t
52
103*
68 68 98 78
on y0 necrotic leaf area
58,* 48 102,** 136,** 90,* 61t 45,* 35 100,** 92,** 53,* 33
99,**
99,**
Mean 52*
38
45,** 56** 14, 5s,* 55,t 29, 79,** 20,44 97,** 55,** 39,t 96**
a Per group in order of increasing inhibitory action in vitro. * P < 0.05, ** P < 0.01, t P < 0.10, levels of significance for the differences values of mycelial development or necrosis with or without saprophytes pollen.
53 97 76 75 40 82 43 55 50 43 64 52
of the related in presence of
the necrotic area. The positive correlation between superficial mycelial growth and necrosis which was found in previous studies [II], explaining the stimulation of infection in presence of pollen, was confirmed. All values for mycelial growth and corresponding necrosis concerning the experiments of Table 3 were arranged
Myceliol
length
(pm/mmz,
log scale)
FIG. 3. The relationship between the development of the superficial mycelium of D. sorokiniana on the leaf and the subsequent necrosis, as influenced by addition of pollen supplemented with cells of yeast-like fungi. Additions: (x), none; (o), pollen 3%; (o), 3% pollen + Aureobasidium; (o), 3% pollen + Cryptococcus; (A), 3% pollen + Sporobolomyces. Encircled marks indicate the mean value of all data from a similar treatment.
202
N. J. Fokkema
in a scatter diagram (Fig. 3). According to Spear-man’s rank correlation test, the correlation between these variables was significant (P
4
The effects on germination of spores of D. sorokmiana, and subsequent deuelo@mt on leaves and agar coated glass slides, caused by adding yeast-like isolates to the pollen used in the inoculum
Treatment
Germination Exp. 1
In vivo D. sorokiniana (D) D + Pollen (DP) ’ DP+Aa 39 DP+C’” 50 DP+S” 30
54 86 82 84 82 Exp.
In vitro D DP DP+A 39 DP+C 50 DP+S 30
(%) Exp. 2
36
77 94 89 91 94
Exp.
67 100 90 126 102
62 138 126 126 130 4
Exp.
3b
101 142 125 132 142
52 80 80 82 84
* P< 0.05, ** P < 0.01, levels DP treatment. a See Table 2. b Two days after inoculation;
Superficial mycelium (v@m2) Exp. 1 Exp. 2
Germ tubes/100 spores Exp. 1 Exp. 2
64 85 76 84 74
4
56 95 100 96 104
of significance
for
all other
3 days after
data
Exp.
the differences
superficial mycelial saprophytic fungal
loo** 2700 125* 50** 1650
125** 1450 325** 275** 575*
Exp.
Exp.
38
2400** 12 000 3900* 6450 6100* with
the results
4
3850** 30 775 650** 4800** 7075** after
the
inoculation.
The same parameters could be studied in vitro on slides covered with a thin layer of water agar [11] and inoculated by spreading drops of similar suspensions as added to leaves. After 2 or 3 days’ incubation at 15 “C in moist Petri dishes, germination and mycelial growth were assessed. The mycelial development was much higher on the agar slides than on the leaves. This might be due to the fact that slides remain wet longer than leaves. On slides and on leaf surfaces, a reduction of the stimulated mycelial development was noticed without any effect of the antagonists on germination. The effect of the antagonists seems to be independent of the leaf, indicating that phytoalexins are probably not involved in the inhibitory effects under study. Analysis
of the inhibition
on agar plates
The considerable differences in antagonistic properties shown by the three groups of the yeast-like fungi, and the differences even within groups which had been recorded in dual culture experiments on agar did not occur on the surfaces of leaves. Inhibition on agar plates is commonly ascribed to production of
PLATE 1. Effect of seeding a spore suspension of D. .rorokininna over a 6-day-old dual culture of the same pathogen and S. mseus. Notice the undisturbed development along the side of the yeast remote from the Drechslera, and thr slight d?vrlopmmt of colonies within thv zonr of inhibition at the side of the yeast only.
Rble
of fungi
against
203
D. sorokiniana
antibiotics by the antagonists. However, competition for nutrients might also be involved [7, 81. Spores of D. sorokiniana were seeded over 6-day-old dual cultures of antagonists and D. sorokiniana. After 6 h incubation at 23 “C, germination was assessed within the zone of inhibition, if present, and along the border of the yeastlike colony remote from D. sorokiniana (Table 5). Within the zones of inhibition TABLE
5
Germination of D. sorokiniana spores seeded ooer agar plates already growing colonies of yeastlike isolates and D. sorokiniana inoculated 6 days before. Germination was assessed within the zone of inhibition (“in zone”) and along the border of theyeast-like colony remotefrom D. soroldniana (“ex zone”)
Yeast-like
isolate
“In
Aureobasidium
No. No. No.
7 39 49
c~ptococcus
No. No. No.
Sporobolomyces
a Zone
of inhibition
Germination zone”
“Ex
(%) zone”
Germ tubes/ “In zone”
100 spores “Ex zone”
77 86 78
7 0 0
90 97 90
43 50 36
6 0 0 -a -’ 14
100 89 94
14
129 117 103
No. 21 No. 30 No. 13
1 0 5
94 90 82
1 0 5
134 122 116
not present.
between all isolates and D. sorokiniana, germination was almost totally inhibited. However, along the opposite border of the yeast-like colonies, in a zone not wider than 1.5 mm, no inhibitory effect was observed. This indicates that the yeast-like isolates did not produce any substance inhibitory to D. sorokiniana on agar plates. Since germination of Drechslera spores on water agar was still 65%, it is unlikely that lack of nutrients is responsible for the very poor germination within the zone of inhibition. It is also unlikely that Drechslera alone could induce the inhibition of germination, as germination along the border of a Drechslera colony (4 cm in diameter) reached 83%. The seeded Drechslera spores developed into sporulating colonies. Even within the zone of inhibition some colonies appeared but only along the side of the yeast-like colony (Plate 1). This suggests that the inhibition originates rather from the side of Drechslera than from the side of the yeast. The zone of inhibition and the reduction of radial growth on agar plates seems to be the result of the production of metabolic products by Drechslera itself, probably induced by the “antagonists”. The kind of antagonism found within this zone of inhibition is unlikely to play a role in the phyllosphere. DISCUSSION It has been shown here that the antagonistic effects of the phyllosphere fungi on rye leaves against D. sorokiniana are not likely to be based on production of antibiotics or induction of phytoalexins. Diem [8] found evidence that the inhibitory effect of C. herbarum, C. cladosporioides and A. pullulans on the germination of D. sorokiniana spores on agar was caused by nutrient competition. An inhibitory effect on germination could not be observed in the study presented here, probably because
204
N. J. Fokkema
of differences in the nutrient requirements of the strains used. However, nutrient competition in the phyllosphere of rye seems an adequate explanation for the reduction of the effect of pollen on infection. This hypothesis is supported by the absence of differences in antagonistic properties shown by the different groups of saprophytic colonizers on the leaf. The ability of the main phyllosphere colonizers of rye to neutralize the stimulating effect of pollen on infection by D. sorokiniuna recorded here, is in agreement with previous suppositions based on field experiments [II]. Under field conditions, in which the mycoflora always consists of a mixture of the groups studied, the antagonistic effect might be even more pronounced. A comparable interference of the saprophytic mycoflora in a pollen effect was found by Warren [20, 211 studying the leaf mycoflora of a flowering crop of Beta uulgaris and infection by Phoma betae. Reducing the phyllosphere flora by fungicides [13, 16, 191 might have serious consequences for anemophilous crops by eliminating the natural buffering mechanism against the pollen effect. Preliminary experiments showed that application of the fungicide benomyl to rye leaves reduced the populations of Cladosporium and Sporobolomyces to about 5% and eliminated the Aureobasidium population, whereas the “White yeasts” were not affected by benomyl. The relative resistance of Drechslera to benomyl [a is of interest, and may cause further enquiry as to the real effects on the leaf surface mycoflora of the use of fungicides. In the host-pathogen combination used, no significant antagonistic effect on the non-stimulated Drechslera infection was found as leaf necrosis was very restricted. Since the antagonists act by reducing the superficial mycelial growth of the pathogen, similar results might be expected in other host-pathogen combinations where superficial mycelial growth is a prerequisite for infection. This suggestion is supported by the investigations of Akai & Kuramoto [1] with a similar combination of rice and C. miyabeanus. Candida, the dominating yeast on rice leaves, reduced the brown leaf spot to 50%, also without inhibition of germination. They also showed, as in the work reported here, that there was no relationship between inhibition on agar and on the leaf surface. McBride [15] found that infection of larch leaves by Meria laricis was reduced by Sporobolomyces only under low nutrient conditions. In our study, nutrients, e.g. pollen, did not prevent the antagonistic action of Sporobolomyces. This may merely reflect differences in the particular host-parasite combination being studied. Though production of antibiotics by A. pullulans and Sporobolomyces is reported [4, 221, their participation in antagonistic effects must not be overestimated. The discrepancy between the ability of one organism to inhibit the growth of another in culture and its ability to function as an antagonist on the plant surface has already been recognized [I, 3, 121. However, it is assumed that effects in vitro were still indicative for a potential antagonistic capacity in vivo [12]. The “re-inoculation” tests performed here (Table 5) reveal that the inhibition zones in dual cultures with D. sorokiniana were not merely caused by antibiotics produced by the presumed antagonists. Similar observations were reached by Huber & Watson [14] working with Typhula idahoensis and several bacteria. So, when studying antagonism, the use of pure cultures on agar plates can be misleading. Thanks are due to Miss J. A. Achterstraat, Mrs B. M. M. Cnossen-van der Meij and
R61e of fungi
against
D. sorokiniana
205
Miss J. E. Hupkes for their cooperation in a part of the investigation. Thanks are also expressed to Dr K. W. Gams and Drs L. Rodrigues de Miranda, staff members of the Centraal Bureau voor Schimmelcultures, for identifying many of the fungi and to Dr K. M. Old for correction of the English text. REFERENCES 1. AKAI, S. & KURAMOTO, T. (1968). Micro-organisms existing on leaves of rice plants and the occurrence of brown leaf spot. Annals of the Phytopathological Society of Japan 34,313-316. 2. ASARF,-NYAKO, A. (1967). The role of the leaf microflora on epidemiology of the northern leaf blight of corn. Dissertation Abstracts 27-B, 4206-4207. 3. BHATT, D. D. & VAUGHAN, E. K. (1963). Inter-relationships among fungi associated with strawberries in Oregon. Phytopathologr 53, 217-220. 4. BAIGENT, N. L. & OGAWA, J. M. (1960). Activity of the antibiotic produced by Pullulariapullulans. Phytopatholopy 50, 82 (Abstr.) . 5. BOLLEN, G. J. & FUCHS, A. (1970). On the specificity of the in vitro and in ho antifungal activity of benomyl. Netherlands Journal of Plant Pathology 76, 299-312. 6. CHLJ-CHOU, M. & PREECE, T. F. (1968). The effect of pollen grains on infections caused by Botrytis cinerea. Annals of Applied Biology 62, 1 l-22. 7. DIEM, H. G. (1969). Microorganismes de la surface des feuilles. II. Interactions entre quelques champignons parasites et divers saprophytes filamenteux de la phyllosphtre de Forge. Bulletin de l’&ole Nationale Superieure Agronomique de JVanxy 11, 12-17. 8. DIEM, H. G. (1969). Microorganismes de la surface des feuilles. III. Effet de la competition nutritive sur la germination des spores d’une souche d’Helminthosporium sativum. Bulletin de L’Bcole Nationale SupLrieure Agronomique de .Nancy 11, 18-25. 9. ELLIS, M. B. (1971). Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 10. FOKKEMA, N. J. (1971). Influence of pollen on saprophytic and pathogenic fungi on rye leaves. In Ecology of Leaf Surface Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 277-282. Academic Press, London. Il. FOKKEU, N. J. (197 1). The effect of pollen in the phyllosphere of rye on colonization by saprophytic fungi and on infection by Helminthosporium sativum and other leaf pathogens. Netherlands Journal of Plant PatholoQ 77, Supplement no. 1. 12. HEWEL, J. VAN DEN (1970). Antagonistic effects of epiphytic micro-organisms on infection of dwarf bean leaves by Altemaria zinnias. Dissertation, Utrecht, Mededeling, No. 84 of the Phytopathologisch Laboratorium “Willie Commelin Scholten”, Baarn, The Netherlands. 13. HISLOP, E. C. & Cox, T. W. (1969). Effects of captan on the non-parasitic microflora of apple leaves. Transactions of the British Mycological So&y 52, 223-235. 14. HUBER, D. M. & WATSON, R. D. (1966). How valid is the agar plate inhibition test for determining antagonism between soil micro-organisms? Phytopathology 56, 882 (Abstr.). 15. MCBRIDE, R. P. (1971). Micro-organism interactions in the phyllosphere of larch. In EEoloQ$of Leaf Surface Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 545-555. Academic Press, London. 16. MCKENZIE, E. H. C. (1971). Seasonal changes in fungal spore numbers in ryegrass-white clover New Zealand Journal of Ap’cultural pasture, and the effects of benomyl on pasture fungi. Research 14, 379-392. 17. NEWHOOK, F. J. (1957). The relationship of saprophytic antagonism to control of Botrytis cinerea Pers. on tomatoes. New ~ealandJourna1 of Science and Technology Section A 38, 473-481. 18. PORTER, Cl. L. (1924). Concerning the characters of certain fungi as exhibited by their growth in the presence of other fungi. American 3ournal of Botany 11, 168-188. 19. SCOTT, M. A. (1971). Studies on the physiology of some leaf saprophytes. In Ecology of Leaf Surfme Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 203-210. Academic Press, London. 20. WARREN, R. C. (1972). The effect of pollen on the fungal leaf microflora of Beta vulgaris L. and on infection of leaves by Phoma betae. .hfetherlands Journal of Plant Pathology 78, 89-98. 21. WARREN, R. C. (1972). Interference by common leaf saprophytic fungi of the development of Phoma betae lesions on sugar beet leaves. Annals of Applied BioloQ 72, 137-144. 22. YAMASAKI, I., SATOMURA, Y. & YAMAMOTO, T. (1951). The red yeast Sporobolomyces. X. AntiChemical Society of Japan 24, diabetic action and fungistatic action, 3. Journal of the Agricultural 39942.
Plant Pathology
(1973)
3, 195-205
The rdle of saprophytic fungi in antagonism against Drechslera sorokiniana (Helminthosporium on agar plates and on rye leaves with pollen
sativum)
N. J. FOKKEMA Phytopathologisch (Accepted
Laboratorium
for publication
“Willie
September
Commelin
&h&m”,
Baarn,
The Netherlands
1972)
About 50 isolates from each of the four dominant groups in the phyllosphere of rye (Se& cereale) were tested for inhibitory effects against Drechslera sorokiniana (Helminthosporium sativum) in agar culture. The most antagonistic fimgus was Aureobasidium pullulanr, followed by the Sporobolomyces spp. group and then the “White yeasts” (mainly CryPfococcur spp.) . There was considerable variation in inhibitory activity within each group on agar. Isolates of Cladosporium spp. did not show any antagonistic effect against Drechslera in vitro. Inoculations of living rye leaves with D. sorokiniana and isolates from each group of saprophytic fungi always resulted in a remarkable reduction of infection in the presence of rye pollen, contrasting with the varying degrees of antagonistic effect observed in agar culture. Development of superficial mycelium of the pathogen on the rye leaf surface and the subsequent severity of necrosis are positively correlated. Both on leaves and on slides coated with agar, it was the enhanced superficial mycelial growth of the pathogen in the presence of pollen which was inhibited by the saprophytes. Germination of the Drechslera spores was not affected.
INTRODUCTION
Antagonistic action by saprophytic fungi against plant pathogens may be based on competition for nutrients, on production of antibiotics or on induction of phytoalexins. The relevant literature was recently reviewed by Van den Heuvel [IL’]. Antagonism of fungi in relation to Helminthosporium diseases of cereal leaves has been studied by Porter [18], Asare-Nyako [Z] and Akai & Kuramoto [I]. Most of the research gave more attention to the inhibition of the pathogen’s development on agar by a great variety of micro-organisms, than to the effect of the natural microflora on the infection. However, Akai & Kuramoto demonstrated that Candida, a yeast dominating on rice leaves, could reduce infection by Cochliobolus miyabeanus by about 50%. Cladosporium, a dominating hyphal fungus in the phyllosphere, appeared to reduce infection of tomatoes and strawberries by Botrytis cinerea [3, 171. Recent studies have revealed that pollen deposited on fruits or leaves of anemophilous plants can stimulate fungal infection [S, IO, II, 20, 24. The objective of the present study was to examine whether this “pollen effect” was reduced by antagonistic activities of the saprophytic mycoflora under field conditions. Previous studies [II] had shown that field inoculations of rye leaves with Helminthosporium sativum just after flowering resulted in a marked increase of infection compared to the infection which occurred after inoculations before flowering. When rye pollen
196
N. J. Fokkema
was added to leaves already bearing a natural pollen deposit, stimulation of infection did not occur. When pollen was added to comparable leaves upon which pollen deposit had been prevented, infection was stimulated. These results were explained [II] by the hypothesis that the natural pollen deposit stimulated an antagonistic population of saprophytes, which reduced infection by H. sativum in the presence of pollen. The saprophytic mycoflora of rye leaves can be separated into four dominant groups: Cladosporium spp., Aureobasidium pullulans (De Bary) Arnaud, the pink yeast Sporobolomyces roseusKluyver & v. Niel and “White yeasts”, mainly C~ptococcus spp. About 50 isolates of each group were tested for antagonistic properties in dual cultures with Drechslera sorokinianat (Sacc.) Subram & Jain (syn.: Helminthosporium sativum Pamm., King & Bakke). Several of the saprophytic isolates were selected, and their effects on infection of leaves by D. sorokiniana were studied. Attempts were made to correlate their antagonistic properties on the leaf surface with those observed in dual cultures. It has previously been suggested that such a correlation may not exist [I, 3, 121. MATERIALS AND METHODS Antagonism on agar plates
The saprophytes were obtained by shaking rye leaves in water, from which isolations of fungi were subsequently made [II]. They were propagated on PDA slants at 23 “C in darkness. The strain of D. sorokiniana, the conidial state of Cochliobolus sativum (Ito & Kuribayashi) Drechsler ex Dastur, was cultured on oatmeal agar slants at 23 “C in darkness.
FIG. 1. Diagram of the mode of inoculation of agar plates with D. sorokiniana and like test organism. Parameters for inhibition are the width of the zone of inhibition the percentage inhibition of radial growth [ 100 x (rl - rs) /rJ.
a yeast(d) and
After culturing for about 2 weeks, a streak (4 cm) of the yeasts or the yeast-like fungus A. pullulans was made on PDA plates (10 ml per Petri dish of 9 cm diameter) as shown in Fig. 1. In the case of the Cladosporium isolates a loopful of the spore suspension was placed 2 cm from the margin of these plate. Opposite the saprophytes, at a distance of at least 3 cm, a loopful of a spore suspension of D. sorokiniana, containing about 20 conidia, was deposited. After incubation for 6 days at 23 “C t Sensu Ellis
[9].
R6le
of fungi
against
D. sorokiniana
197
in darkness, inhibition of the pathogen’s development was assessed by two parameters, viz. the percentage of inhibition of radial growth [lo0 x (rr-rs)/rJ and the width of the zone of inhibition measured at the smallest distance between both colonies (d), as illustrated in Fig, 1. The use of two parameters allows discrimination between inhibition of the growth of the pathogen and the type of interaction, such as inhibiMoreover, the various growth rates of the saprotion at a distance or by contact. phytes will affect the radial growth of the pathogen independent of their antagonistic action, whereas the zone of inhibition remains unaffected. On the other hand, the width of the zone of inhibition may be affected also by retardation of the growth of the saprophyte. Antagonism
on rye leaves
Summer rye, Secale cereale L. cv. “Petkuser”, was used as the host and grown as described earlier [II]. The penultimate leaves were inoculated with a suspension consisting of I.5 to 3.5 x lo5 spores of D. sorokiniana, 3 x 10’ spores (cells) of an isolate of the yeasts or A. pullulans or l-25 x 10’ spores of a Cladosporium isolate, and 30 mg of rye pollen per 1 ml of O*1o/o Tween 80. As a control, leaves were inoculated with D. sorokiniana alone, with or without pollen addition. Generally, a set of eight leaves received the same treatment. The fungi were cultured similarly as for the in vitro studies. Yeasts and Aureobasidium, however, were grown for 6 days instead of 2 weeks. Spores and yeast cells were suspended in O-1o/0 Tween 80 by scraping the cultures with an inoculation needle followed by shaking. Masses of agar and large mycelial fragments were removed by filtering through glasswool. Before inoculation, the leaves were drawn between the moistened thumb and index finger to disturb the wax layer ensuring a uniform distribution of the inoculum drop. Then 0.1 ml of the inoculation suspension was pipetted on the leaf base and spread over the whole surface by drawing the leaf between thumb and index finger. Immediately after inoculation the plants were placed in large transparent plastic covered cages lined with moist filter paper. The temperatures varied between 18 and 25 “C and relative humidity from 85 to 95%. The areas of necrosis were assessed 7 days after inoculation by means of a planimeter and expressed as a percentage of the leaf surface area. In some experiments, the pre-infection development of the pathogen was assessed 3 days after inoculation. Therefore additional sets of eight leaves were inoculated. On these leaves spore germination and the development of superficial mycelium on leaf parts, 6 to 9 cm from the base of the lamina, were studied on transparent adhesive tape replicas [II.] The superficial mycelial development was assessed by projecting photomicrographs from the replica on to a paper. Photomicrographs were made from three distinct areas on each replica (total area of about 0.8 mm2 per replica). The length of the hyphae was measured with a curvimeter and expressed in units of 25 pm/mm2 of leaf surface. The remaining parts of the leaves, used for the replicas, were used for the assessment of the development of the added saprophytes by plating out the washing water [II]. The capacity of the saprophytes to neutralize the pollen effect was expressed as the relative inhibitory effect (R.I.E.), which may be calculated by 100 x (DP DPS)/(DPD), where DP, DPS and D are the results after inoculation with Drechslera plus pollen (DP), with DP plus a saprophyte (DPS) and with Drechslera
N. J. Fokkema
198
alone (D). Either the effect of the saprophytes on the superficial mycelial development of the pathogen or on the necrosis was expressed as the R.I.E. The data from each of the experiments were subjected to the Wilcoxon’s two sample test. RESULTS Antagonism
on agar plates The behaviour of the 48 Cladosporium isolates, mostly belonging to C. herbarum (Pers.) Link ex Fr., against D. sorokiniana, was very uniform. Both colonies grew undisturbed until they came in contact with each other. Their mycelia did not intermingle. 1
I
I
I
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,
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(b)
7 40
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-Fe
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3-
g
2-
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,-
-
t
.*
I 50
60
Inhibition of radio1 growth
.: l
.
i.
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-3
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t 20
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I 30
Inhibition of radio1 growth
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-2-
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Inhibition of radial growth
I 30 (%I
FIG. 2. Antagonistic properties of isolates of Aureobmidium (a), Sporobolomyces (b) and “White yeasts” (c) against D. s~okiniana in dual cultures, expressed as the width of the zone of inhibition and the percentage inhibition of radial growth of D. sorokiniana. + indicates the isolates used in in viva experiments.
R61e of fungi
against
199
D. sorokiniana
The behaviour of the yeasts and of the yeast-like fungus A. pullu&s was much more variable. These experiments were repeated three times. The largest time interval between replicates was at least 4 months, which may give an indication of the stability of the antagonistic properties of a particular isolate over this period. The mean results for each isolate are given in Fig. 2. Nearly all isolates of the A. pullulans group caused inhibition at a distance. The inhibition of the radial growth, as compared with the zone of inhibition, was more pronounced than in the yeasts, but this was mainly due to the fact that their outgrowth was much less than that of A. pullulans. Unlike the yeasts, the development of some isolates of A. pullulans towards the Drechslera colony was inhibited too, indicating mutual inhibition. About 70% of the S’orobolomyces isolates caused inhibition at a distance, others could be approached by Drechslera until contact was made, or were even overgrown by it, resulting in a negative value for the distance between the colonies. Only 30% of “White yeasts” caused inhibition at a distance, the remaining isolates were overgrown. Forty-four per cent of the isolates inhibited the radial growth by 20% or less. The range between the three values for d and 100 x (rl-rs)/r]. of the individual isolates in the three experiments gives an impression of the reproducibility of the antagonistic properties in vitro. The mean value of these ranges concerning all isolates of A. pullulans amounted to 1.5 mm and to 8*Oo/o for the distance and for the percentage inhibition of radial growth respectively. Concerning the isolates of Sporobolomyces, these figures were 4.3 mm and 13*5%, while for the “White yeasts” the means of the ranges were 3.9 mm and 16.3%. Considering these values and the scales used for both parameters in Fig. 2, it may be concluded that the reproducibility of the antagonistic properties in vitro of a single isolate is slight. Antagonism
on leaves
The isolates of C. herbarum did not show any antagonistic effect to D. sorokiniana in vitro. Two isolates were taken to investigate the effect of C. hzrbarum on the infection of rye leaves by D. sorokiniana, in the presence of pollen (Table 1). The infection, TABLE Effect of adding C. herbarum
Treatment
1
isolates to the inoculum on the stimulatoly of rye leaves by D. sorokiniana
Relative inhibitory
Mean necrotic leaf area (%) Exp. 1 Exp. 2 Exp. 3
D. sorokiniana (D) D + Pollen (DP) DP + Clad. 42 DP f Clad. 33
3** 57 10** 36*
* P< 0.95, ** P< O*Ol, the DP treatment.
levels
1** 72 36* 42** of significance
Exp.
6** 64 17** 6** for
the
effect of pollen on infection
1
87 39 differences
Exp.
2
50 42 with
the results
effect Exp.
3
82 100 after
which is normally low, was highly stimulated when pollen was added. Addition of Cladosporium spores reduced the pollen effect to a considerable degree. Both isolates behaved similarly, and a third C. herbarum isolate, used in a single experiment, gave the same results. Three days after inoculation, the addition of Cladosporium had
200
N. J. Fokkema
resulted in a colonization varying from 24 000 to 67 000 propagules of Cladosporium per cm2 of leaf surface, as assessed by plating out the washing water. The superficial growth of mycelium of D. sorokiniana could not be measured because it was impossible to distinguish it from mycelium of C. herbarum. For comparison of the antagonistic effects of the yeast-like fungi in vitro and in vivo, three isolates of each group were selected for in vivo studies. These isolates, already indicated in Fig. 2, represented the most inhibitory, the moderate or the least inhibitory isolates (Table 2). The experiments were set up in the same way as those with Cladosporium. In most experiments, the superficial mycelial development of Antagonistic
properties
against
D. sorokiniana Zone d (mm)
Isolatesa Aureobasidium
No. No. No.
7 39 49
1 3 5.3
c~ptococcus
No. No. No.
43 50 36
-9-5 -0.7 1.5
Sporobolomyces
No. No. No.
21 30 13
-2
TABLE 2 on agar of yeast-like
of inhibition Range (mm)
3.3 443
0 8 1 2 2.5 6 5 1.5
saprophytic
fungal
isolates
Inhibition on radial growth (%) 100x (rl-r2)/rl Range (%) 52 50 54
13 10 6
8 20 30
7 10 2
21 35 37
17 6 17
a A.7, A.39, A.49 were identified as -4. pullulans (De Bary) Arnaud; (Kuff.) Skinner var. laurentii; C.50 as C. laurentii var. javescens Lodder albidw (Saito) Skinner var. d#lums (Zach) Phaff et Fell; S.21, S.30, as S. roseus Kluyver et V. Niel.
C.43 as C. laurentii et V. Rij; C.36 as C. S.13 were identified
was also determined. The results are presented as values of the relative effect (Table 3). On average the isolates of Aureobasidium, Cryptococcus and S’orobolomyces reduced the stimulation of the infection induced by pollen by a half. The mycelial development of D. sorokiniana was affected to a comparable degree. Contrary to the effects in vitro, no significant difference was found between the general antagonistic action of the three groups, nor in the antagonistic action of the selected isolates within a group. Three days after inoculation, the addition of yeast-like micro-organisms resulted in colonization varying from 30 000 to 600 000 cells of these micro-organisms per cm2 of leaf surface. Within these limits no effect of the concentration of the antagonist on its action could be noticed. In general the addition resulted in about 180 000 cells per cm2. Such a density can be reached under favourable conditions after flowering in the field before yellowing [II]. Th e colonization of the leaves to which suspensions of micro-organisms had not been added seldom reached 1 o/0of the colonization of leaves inoculated with saprophytes. D. sorokiniana
inhibitory
Analysis
of
the reduction of the pollen efect
The results of Table 3 strongly suggest that the reduction of infection was caused by a corresponding reduction of the mycelial growth over the leaf surface. Such a reduction might diminish the number of penetration sites, and consequently reduce
Rale
of fungi
against
201
Lhorokiniana TABLE
3
Effect on subsequent infection of adding yeast-like components of the phyllosphere Jora to pollen and D. sorokiniana on rye leaves. Reduction of the ‘tpollen effect” is expressed as the relative inhibitory effect (R.I.E.), calculated for the mean results of several exjeriments Addition of micro-organisms? Aureobasidium
cryptococcus
Sporobolomyces
mycelial
R.I.E. on development
No. 7 No. 39 No. 49 Mean
6,72* loo,* 85** 55,117**
No. 43 No.50 No.36 Mean
45,49 lo&** 71,**
No. 21 No. 30
66,70 41, 67,*
No. 13 Mean
91,*
39 92 86 72 47 96 58 67
89** 46
90,t
R.I.E. Mean
89,*
92,t
52
103*
68 68 98 78
on y0 necrotic leaf area
58,* 48 102,** 136,** 90,* 61t 45,* 35 100,** 92,** 53,* 33
99,**
99,**
Mean 52*
38
45,** 56** 14, 5s,* 55,t 29, 79,** 20,44 97,** 55,** 39,t 96**
a Per group in order of increasing inhibitory action in vitro. * P < 0.05, ** P < 0.01, t P < 0.10, levels of significance for the differences values of mycelial development or necrosis with or without saprophytes pollen.
53 97 76 75 40 82 43 55 50 43 64 52
of the related in presence of
the necrotic area. The positive correlation between superficial mycelial growth and necrosis which was found in previous studies [II], explaining the stimulation of infection in presence of pollen, was confirmed. All values for mycelial growth and corresponding necrosis concerning the experiments of Table 3 were arranged
Myceliol
length
(pm/mmz,
log scale)
FIG. 3. The relationship between the development of the superficial mycelium of D. sorokiniana on the leaf and the subsequent necrosis, as influenced by addition of pollen supplemented with cells of yeast-like fungi. Additions: (x), none; (o), pollen 3%; (o), 3% pollen + Aureobasidium; (o), 3% pollen + Cryptococcus; (A), 3% pollen + Sporobolomyces. Encircled marks indicate the mean value of all data from a similar treatment.
202
N. J. Fokkema
in a scatter diagram (Fig. 3). According to Spear-man’s rank correlation test, the correlation between these variables was significant (P
4
The effects on germination of spores of D. sorokmiana, and subsequent deuelo@mt on leaves and agar coated glass slides, caused by adding yeast-like isolates to the pollen used in the inoculum
Treatment
Germination Exp. 1
In vivo D. sorokiniana (D) D + Pollen (DP) ’ DP+Aa 39 DP+C’” 50 DP+S” 30
54 86 82 84 82 Exp.
In vitro D DP DP+A 39 DP+C 50 DP+S 30
(%) Exp. 2
36
77 94 89 91 94
Exp.
67 100 90 126 102
62 138 126 126 130 4
Exp.
3b
101 142 125 132 142
52 80 80 82 84
* P< 0.05, ** P < 0.01, levels DP treatment. a See Table 2. b Two days after inoculation;
Superficial mycelium (v@m2) Exp. 1 Exp. 2
Germ tubes/100 spores Exp. 1 Exp. 2
64 85 76 84 74
4
56 95 100 96 104
of significance
for
all other
3 days after
data
Exp.
the differences
superficial mycelial saprophytic fungal
loo** 2700 125* 50** 1650
125** 1450 325** 275** 575*
Exp.
Exp.
38
2400** 12 000 3900* 6450 6100* with
the results
4
3850** 30 775 650** 4800** 7075** after
the
inoculation.
The same parameters could be studied in vitro on slides covered with a thin layer of water agar [11] and inoculated by spreading drops of similar suspensions as added to leaves. After 2 or 3 days’ incubation at 15 “C in moist Petri dishes, germination and mycelial growth were assessed. The mycelial development was much higher on the agar slides than on the leaves. This might be due to the fact that slides remain wet longer than leaves. On slides and on leaf surfaces, a reduction of the stimulated mycelial development was noticed without any effect of the antagonists on germination. The effect of the antagonists seems to be independent of the leaf, indicating that phytoalexins are probably not involved in the inhibitory effects under study. Analysis
of the inhibition
on agar plates
The considerable differences in antagonistic properties shown by the three groups of the yeast-like fungi, and the differences even within groups which had been recorded in dual culture experiments on agar did not occur on the surfaces of leaves. Inhibition on agar plates is commonly ascribed to production of
PLATE 1. Effect of seeding a spore suspension of D. .rorokininna over a 6-day-old dual culture of the same pathogen and S. mseus. Notice the undisturbed development along the side of the yeast remote from the Drechslera, and thr slight d?vrlopmmt of colonies within thv zonr of inhibition at the side of the yeast only.
Rble
of fungi
against
203
D. sorokiniana
antibiotics by the antagonists. However, competition for nutrients might also be involved [7, 81. Spores of D. sorokiniana were seeded over 6-day-old dual cultures of antagonists and D. sorokiniana. After 6 h incubation at 23 “C, germination was assessed within the zone of inhibition, if present, and along the border of the yeastlike colony remote from D. sorokiniana (Table 5). Within the zones of inhibition TABLE
5
Germination of D. sorokiniana spores seeded ooer agar plates already growing colonies of yeastlike isolates and D. sorokiniana inoculated 6 days before. Germination was assessed within the zone of inhibition (“in zone”) and along the border of theyeast-like colony remotefrom D. soroldniana (“ex zone”)
Yeast-like
isolate
“In
Aureobasidium
No. No. No.
7 39 49
c~ptococcus
No. No. No.
Sporobolomyces
a Zone
of inhibition
Germination zone”
“Ex
(%) zone”
Germ tubes/ “In zone”
100 spores “Ex zone”
77 86 78
7 0 0
90 97 90
43 50 36
6 0 0 -a -’ 14
100 89 94
14
129 117 103
No. 21 No. 30 No. 13
1 0 5
94 90 82
1 0 5
134 122 116
not present.
between all isolates and D. sorokiniana, germination was almost totally inhibited. However, along the opposite border of the yeast-like colonies, in a zone not wider than 1.5 mm, no inhibitory effect was observed. This indicates that the yeast-like isolates did not produce any substance inhibitory to D. sorokiniana on agar plates. Since germination of Drechslera spores on water agar was still 65%, it is unlikely that lack of nutrients is responsible for the very poor germination within the zone of inhibition. It is also unlikely that Drechslera alone could induce the inhibition of germination, as germination along the border of a Drechslera colony (4 cm in diameter) reached 83%. The seeded Drechslera spores developed into sporulating colonies. Even within the zone of inhibition some colonies appeared but only along the side of the yeast-like colony (Plate 1). This suggests that the inhibition originates rather from the side of Drechslera than from the side of the yeast. The zone of inhibition and the reduction of radial growth on agar plates seems to be the result of the production of metabolic products by Drechslera itself, probably induced by the “antagonists”. The kind of antagonism found within this zone of inhibition is unlikely to play a role in the phyllosphere. DISCUSSION It has been shown here that the antagonistic effects of the phyllosphere fungi on rye leaves against D. sorokiniana are not likely to be based on production of antibiotics or induction of phytoalexins. Diem [8] found evidence that the inhibitory effect of C. herbarum, C. cladosporioides and A. pullulans on the germination of D. sorokiniana spores on agar was caused by nutrient competition. An inhibitory effect on germination could not be observed in the study presented here, probably because
204
N. J. Fokkema
of differences in the nutrient requirements of the strains used. However, nutrient competition in the phyllosphere of rye seems an adequate explanation for the reduction of the effect of pollen on infection. This hypothesis is supported by the absence of differences in antagonistic properties shown by the different groups of saprophytic colonizers on the leaf. The ability of the main phyllosphere colonizers of rye to neutralize the stimulating effect of pollen on infection by D. sorokiniuna recorded here, is in agreement with previous suppositions based on field experiments [II]. Under field conditions, in which the mycoflora always consists of a mixture of the groups studied, the antagonistic effect might be even more pronounced. A comparable interference of the saprophytic mycoflora in a pollen effect was found by Warren [20, 211 studying the leaf mycoflora of a flowering crop of Beta uulgaris and infection by Phoma betae. Reducing the phyllosphere flora by fungicides [13, 16, 191 might have serious consequences for anemophilous crops by eliminating the natural buffering mechanism against the pollen effect. Preliminary experiments showed that application of the fungicide benomyl to rye leaves reduced the populations of Cladosporium and Sporobolomyces to about 5% and eliminated the Aureobasidium population, whereas the “White yeasts” were not affected by benomyl. The relative resistance of Drechslera to benomyl [a is of interest, and may cause further enquiry as to the real effects on the leaf surface mycoflora of the use of fungicides. In the host-pathogen combination used, no significant antagonistic effect on the non-stimulated Drechslera infection was found as leaf necrosis was very restricted. Since the antagonists act by reducing the superficial mycelial growth of the pathogen, similar results might be expected in other host-pathogen combinations where superficial mycelial growth is a prerequisite for infection. This suggestion is supported by the investigations of Akai & Kuramoto [1] with a similar combination of rice and C. miyabeanus. Candida, the dominating yeast on rice leaves, reduced the brown leaf spot to 50%, also without inhibition of germination. They also showed, as in the work reported here, that there was no relationship between inhibition on agar and on the leaf surface. McBride [15] found that infection of larch leaves by Meria laricis was reduced by Sporobolomyces only under low nutrient conditions. In our study, nutrients, e.g. pollen, did not prevent the antagonistic action of Sporobolomyces. This may merely reflect differences in the particular host-parasite combination being studied. Though production of antibiotics by A. pullulans and Sporobolomyces is reported [4, 221, their participation in antagonistic effects must not be overestimated. The discrepancy between the ability of one organism to inhibit the growth of another in culture and its ability to function as an antagonist on the plant surface has already been recognized [I, 3, 121. However, it is assumed that effects in vitro were still indicative for a potential antagonistic capacity in vivo [12]. The “re-inoculation” tests performed here (Table 5) reveal that the inhibition zones in dual cultures with D. sorokiniana were not merely caused by antibiotics produced by the presumed antagonists. Similar observations were reached by Huber & Watson [14] working with Typhula idahoensis and several bacteria. So, when studying antagonism, the use of pure cultures on agar plates can be misleading. Thanks are due to Miss J. A. Achterstraat, Mrs B. M. M. Cnossen-van der Meij and
R61e of fungi
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Miss J. E. Hupkes for their cooperation in a part of the investigation. Thanks are also expressed to Dr K. W. Gams and Drs L. Rodrigues de Miranda, staff members of the Centraal Bureau voor Schimmelcultures, for identifying many of the fungi and to Dr K. M. Old for correction of the English text. REFERENCES 1. AKAI, S. & KURAMOTO, T. (1968). Micro-organisms existing on leaves of rice plants and the occurrence of brown leaf spot. Annals of the Phytopathological Society of Japan 34,313-316. 2. ASARF,-NYAKO, A. (1967). The role of the leaf microflora on epidemiology of the northern leaf blight of corn. Dissertation Abstracts 27-B, 4206-4207. 3. BHATT, D. D. & VAUGHAN, E. K. (1963). Inter-relationships among fungi associated with strawberries in Oregon. Phytopathologr 53, 217-220. 4. BAIGENT, N. L. & OGAWA, J. M. (1960). Activity of the antibiotic produced by Pullulariapullulans. Phytopatholopy 50, 82 (Abstr.) . 5. BOLLEN, G. J. & FUCHS, A. (1970). On the specificity of the in vitro and in ho antifungal activity of benomyl. Netherlands Journal of Plant Pathology 76, 299-312. 6. CHLJ-CHOU, M. & PREECE, T. F. (1968). The effect of pollen grains on infections caused by Botrytis cinerea. Annals of Applied Biology 62, 1 l-22. 7. DIEM, H. G. (1969). Microorganismes de la surface des feuilles. II. Interactions entre quelques champignons parasites et divers saprophytes filamenteux de la phyllosphtre de Forge. Bulletin de l’&ole Nationale Superieure Agronomique de JVanxy 11, 12-17. 8. DIEM, H. G. (1969). Microorganismes de la surface des feuilles. III. Effet de la competition nutritive sur la germination des spores d’une souche d’Helminthosporium sativum. Bulletin de L’Bcole Nationale SupLrieure Agronomique de .Nancy 11, 18-25. 9. ELLIS, M. B. (1971). Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. 10. FOKKEMA, N. J. (1971). Influence of pollen on saprophytic and pathogenic fungi on rye leaves. In Ecology of Leaf Surface Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 277-282. Academic Press, London. Il. FOKKEU, N. J. (197 1). The effect of pollen in the phyllosphere of rye on colonization by saprophytic fungi and on infection by Helminthosporium sativum and other leaf pathogens. Netherlands Journal of Plant PatholoQ 77, Supplement no. 1. 12. HEWEL, J. VAN DEN (1970). Antagonistic effects of epiphytic micro-organisms on infection of dwarf bean leaves by Altemaria zinnias. Dissertation, Utrecht, Mededeling, No. 84 of the Phytopathologisch Laboratorium “Willie Commelin Scholten”, Baarn, The Netherlands. 13. HISLOP, E. C. & Cox, T. W. (1969). Effects of captan on the non-parasitic microflora of apple leaves. Transactions of the British Mycological So&y 52, 223-235. 14. HUBER, D. M. & WATSON, R. D. (1966). How valid is the agar plate inhibition test for determining antagonism between soil micro-organisms? Phytopathology 56, 882 (Abstr.). 15. MCBRIDE, R. P. (1971). Micro-organism interactions in the phyllosphere of larch. In EEoloQ$of Leaf Surface Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 545-555. Academic Press, London. 16. MCKENZIE, E. H. C. (1971). Seasonal changes in fungal spore numbers in ryegrass-white clover New Zealand Journal of Ap’cultural pasture, and the effects of benomyl on pasture fungi. Research 14, 379-392. 17. NEWHOOK, F. J. (1957). The relationship of saprophytic antagonism to control of Botrytis cinerea Pers. on tomatoes. New ~ealandJourna1 of Science and Technology Section A 38, 473-481. 18. PORTER, Cl. L. (1924). Concerning the characters of certain fungi as exhibited by their growth in the presence of other fungi. American 3ournal of Botany 11, 168-188. 19. SCOTT, M. A. (1971). Studies on the physiology of some leaf saprophytes. In Ecology of Leaf Surfme Micro-organisms (T. F. Preece & C. H. Dickinson, eds), pp. 203-210. Academic Press, London. 20. WARREN, R. C. (1972). The effect of pollen on the fungal leaf microflora of Beta vulgaris L. and on infection of leaves by Phoma betae. .hfetherlands Journal of Plant Pathology 78, 89-98. 21. WARREN, R. C. (1972). Interference by common leaf saprophytic fungi of the development of Phoma betae lesions on sugar beet leaves. Annals of Applied BioloQ 72, 137-144. 22. YAMASAKI, I., SATOMURA, Y. & YAMAMOTO, T. (1951). The red yeast Sporobolomyces. X. AntiChemical Society of Japan 24, diabetic action and fungistatic action, 3. Journal of the Agricultural 39942.