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The effects of fungicides on soil fungal populations
So,/ Bwl
B,ochum. Vol 1I. pp. 291to303 0 PergamonPress Ltd 1979. Pnnted m Great Br~tam
0038.0717/79'0601-029750200/0
THE EFFECTS OF FUNGICIDES FUNGAL POPULATIONS A. J. Department
of Biological
and G.J.
KUTHUBUTHEEN*
Sciences,
University
(Accrprrci
of Aston,
3 NoNmhrr
F.
Gosta
ON SOIL
PUGH
Green,
Birmingham,
B4 7ET.
197X)
Summary-Four fungicides, Captan. Dicloran, Thiram and Verdasan were applied at 2%day intervals for 12 consecutive months and their effects on soil fungal numbers and the incidence of individual species were studied. Immediately after application, these fungicides reduced the number of fungal compared with control. Captan- and Dicloranpropagules in soil by 23, I I, 36 and 50% respectively treated soils were rapidly recolonised within 7 days of the application of fungicides. The effects of Thiram and Verdasan were more persistent: the fungal numbers in soils treated with these fungicides did not recover sufficiently to reach control levels throughout the sampling period. Fewer species of fungi were isolated from Thiram- and Verdasan-treated soils than from Captan- and Dicloran-treated soils. Chrysosporium pannorum (Link) Hughes was the principal species isolated from Verdasan-treated
soil and the fungus was isolated in increased numbers immediately after application of Verdasan than on subsequent sampling days. Cladosporium cladosporioides (Fres.) de Vries, Morrierella minurissima van Tiegh, Trichocladium asperum Harz, Trichoderma humarum (Bon.) Bain. and Zyyorhynchus moelleri Vuill. were found to be generally tolerant of all four fungicides. However, Bofryotrichum piluliferum Sacc. and Marchal, Gliocladium roseurn Bain., Humicola @co-atra Traaen, Sepedonium chrysospermum (Bull.) Link ex Fr., and Trichodermn oiride Pers. ex Fr. were generally intolerant of the fungicides but rapidly recolonised the treated soils. While the concentrations of Captan and Dicloran used were fungistatic to 7: ciride, Thiram and Verdasan were fungicidal.
MATERIALS
INTRODUCTION
The changes in the fungal population of soils treated with fungicides may be due to the direct effect of the chemicals on soil fungi. Because most cellular processes are common to all fungi, fungicides kill or adversely affect both the target fungi and a wide range of non-target species (Domsch, 1964, 1970; Kreutzer, 1965; Wainwright, 1977). The changes in the fungal population of treated soils may also, however, be due to the alteration of the physico-chemical characteristics of the soil by fungicides. The application of the organo-mercurial fungicide Verdasan, for example, was shown to reduce decomposition and contribute to an accumulation of organic matter (Pugh and Williams, 1971). This in turn could result in waterlogging and anaerobic conditions which would further reduce microbial activity. Pugh and Williams (1971) studied the fungal populations of a golf-course in Nottingham to which Verdasan had been added twice a year since 1953. Wainwright and Pugh (1975) followed the changes in fungal populations after a single application of some commonly used fungicides. Our aim was to explore the changes brought about by successive monthly applications of fungicides and in doing so, to link the earlier studies. The long-term effects of the fungicides on the soil mycoflora were monitored to determine successional patterns and to distinguish between tolerant and recolonising species.
* Present address: Department of Botany. Malaya, Kuala Lumpur 22-11, Malaysia. S.RB,1,3--P
University
of
AND
METHODS
An 8 x 8 m latin square plot was laid out on a grassland site at the University of Nottingham. To facilitate spraying and to prevent cross-contamination, the plot was divided into 25 sub-squares (1 x 1 m), each bounded by a path 0.5 m. The soil was a sandy-loam with a mean pH 6.8; water holding capacity, 34.7 ml lOOg_-’ soil; loss of weight on ignition, 2.72”;; and total N, 0.169/,. Starting from 9 February 1975. the plots were sprayed at 28-day intervals with the following fungicides: Captan- a.i. SOY/;;(w/w) N-trichloromethylthio-4-cyclohexene-1.2,dicarboximide; Dicloran-a.i. 4”,‘, (w/w) 2,6-dichloro-4-nitroaniline; Thiram-a.i. 501;, (w/w) tetramethylthiuram disulphide and Verdasana.i. 2.5”~; (w/w) organically combined mercury. Each fungicide treatment occurred once in each row and once in each column of the plot. The five untreated squares served as control and were sprayed with tapwater. The fungicides were applied at the following rates: Captan 9 kg ha-‘; Dicloran 2 kg ha-‘; Thiram 6.7 kg ha- ’ ; and Verdasan 12.5 kg ha- I. Appropriate amounts of the fungicides were mixed in the same volume of water. Using random sampling numbers (Fisher and Yates, 1963). soil cores (2-3 cm deep) were taken immediately after spraying and subsequently after 7, 14 and 28 days. The samples taken immediately after spraying were designated Day 0 samples. A total of 48 such samples (including 12 immediately after spraying) were taken from both the control and treated plots. The soil samples from the 5 replicate plots of each treatment were mixed and the fungal 297
A. J.
298
KUTHURUTHEEN and G. .I F’. PN;H
numbers were assessed within 24 h of collection. The seven soil samples from Day 2X of the November 1975 cycle to Day 0 of the January 1976 cycle, however. were stored at 4,C and plated out subsequently. The dilution method was used to assess the gross number of fungal propagules present in soil while the soil plate method (Warcup, 1950) was used to determine the frequency and occurrence of species. Cellulose agar (Eggins and Pugh, 1962) was used for both methods. The presence of a species on one of the five replicate soil plates was considered as one isolation with a frequency of 2(X,,. The plates were incubated at 25 C and examined after 7 days and subsequently at regular intervals until no new colonies or species were observed. RESULTS
The changes in the number of propagules in soils treated with the fungicides are shown in Figs la-b. In the control plots, lowest numbers of fungi were obtained from about December to March while there was a general abundance in the other months. This pattern of occurrence was not altered in Captan- and Dicloran-treated soils. The application of Thiram and Verdasan. however. suppressed the peaks of occurrence of fungi in the treated soils. The fungal counts obtained with Day 0 samples of the fungicide-treated soils were generally lower than those obtained with control and Day 7, 14 and 28 samples. The mean numbers of prop~lgules in the Day 0 samples of C’aptan-. Thiram. and Verdasan-treated soils from February to October were significantly lower compared with control. (Fig. 2). On Days 7. 14 and 2X. however, the fungal numbers in the Cap-
Fig. 1b. The effects of Dicloran and Thiram on the mean number of hrngal propagules in soil from FcbOct, B Thlram: l Control: 0 0 Dicforan: 0 0 7 funglcidc application.
tan-treated soil increased to be comparable with that of control. The etfccts of Thiran and Verdasan were more persistent; the fungal numhcrs in soils trcatcd with these fungicides did not recover sufficiently to be comparable with control numhcrs. The mean fungal numbers in Dicloran-treated soil on all four sampling periods were not significantly lower than that of the control soil. In general. the fungal numbers were reduced most by Verdasan, then by Thiram. Captan and Dicloran in that order. The reductions in the mean number of propagules after application of Captan. & -’ soil immediately Thiram and Verdasan were significant compared with control (Table 1). Mowever. in the Day 7. 14 and 2X samples, the reductions in mean numbers of propagules from only the Thiram- and Verdasan-treated soils remained significant. Soil plute method
Fig. la. The etl’ects of Captan and Verdasan on the mean of fungal propagules in soil from Feb-Oct. Control: A----- a Captan; 0-n Verdasan;
number 0 -*
1 fungicide
application.
The results obtained with the soil plate method were comparable with those observed with the dilution method. In the control soils, lower number of isolations were obtained from about December to March white there was a general abundance in the other months. While these periods of peak isolations were evident in the Captan- and Dicloran-treated soils, in the Thiram- and Verdasan-treated soils the periods of general abundance were absent. The mean number of isolations obtained from Thiram- and Verdasan-treated soils throughout the study did not recover suthciently to be comparable with control (Fig. 3). The mean number of isolations from Captan- and Dicloran-treated soils were significantly lower than control on Day 0. Subsequently, the soils treated with these fungicides were rapidly recolonised so that the fungal numbers were comparable with control.
299
Fungicides and soil fungi
l-
>-
b
0
14
I
Sampling
20
period (days)
Fig. 2. The mean number of propagules on Day 0, 7, 14 and 28 in control and treated soils from Thiram; 0-O Verdasan. FebOct. O---O Control; A---A Captan; o--<) Dicloran; (Vertical bars show 95% confidence limits.)
The reductions in the mean number of isolations immediately after application of all four fungicides were significant compared with control (Table 2). However in the Day 7, 14. and 28 samples, the reductions in the mean number of isolations from only the Thiram- and Verdasan-treated soils remained significant. The periods of peak occurrence for the number of propagules and for the number of isolations in the control soil corresponded to the period when the maximum number of species were isolated from the soil plates. Generally, immediately after application of the fungicides, fewer species of fungi were isolated from the fungicide-treated soils than from control soils. The effects of Thiram and Verdasan were more deleterious; only 5-7 species were consistently isolated immediately after application of the fungicides (Table 3). The mean number of isolations and species were reduced most by Verdasan, then by Thiram, Captan and Dicloran, in that order. Cladosporium cladosporioides, Mortierella minutissima, Trichocladium asperum, Trichoderma hamatum and Zygorhynchus moelleri were found to be generally
tolerant of all four fungicides. These fungi were isolated in comparable frequencies both immediately
after spraying and on subsequent sampling days. However, Botryotrichum piluliferum, Gliocladium roseum, Humicola fisco-atra, Sepedonium chrysospermum and Trichoderma viride were generally intolerant
of the fungicides. These fungi were either absent or were isolated only in low frequencies immediately after application. But subsequently the fungi rapidly recolonized the treated soils and were therefore identified as the recolonizer species. The principal recolonizing fungi of Thiram-treated soils were Penicillium spp. and 7: viride. Of the species isolated, Apiosordaria uerruculosa (Jensen) von Arx and Gams, was isolated throughout the year in control soils, with a minimum occurrence between August and September. It was tolerant of Captan, Dicloran and Thiram and was isolated on Day 0 from soils treated with these fungicides. Its numbers tended to increase on subsequent sampling days. A. verruculosa, however, was not isolated on any of the Day 0 samples of Verdasan-treated soil. It occurred in Verdasan-treated soils only in the winter months on Days 7, 14 and 28. Botryotrichum piluliferum was isolated throughout the year in control soils with a general abundance in the summer and autumn months. In Day 0 samples
A, J.
300
KUTHUBUTHEEN and
Sampling Fig. 3. The mean number Feb-Oct. O-,--• Control;
Table
1. Reduction
$0 (days)
in the mean number of propagules g-’ percentage of control
2. Reduction
soil as a
Day 0
7
14
28
23* II 36* 50*
12 5 32* 42*
7 I 26* 33*
(t0.3) 0.8 22* 29s
* Significantly different compared with control.
Captan Dicloran Thiram Verdasan
period
of isolations on Day 0. 7, 14 and 28 from 45 replicate soil plates from Dicloran; 0-0 Thiram; U----U Verdasan. A--A Captan; (Vertical bars show 957; confidence limits.)
Captan Dicloran Thiram Verdasan
Table
PUGH
A
i
i
G. J. F.
from control
P = 0.05; _t indicates
in the mean number of control
of isolations
an increase
as a percentage
Day 0
7
14
28
31* 21’ 41* 55*
14* 6 25* 41*
c+:.5, 20* 31*
7 3 12* 23*
* Si~i~cantiy different compared with control.
from control
of Captan-treated soil, B. pilulifirurn was either ehminated or isolated only at reduced frequencies. While its numbers were not markedly reduced in Diclorantreated soil, in Thiram and Verdasan-treated soils, I?. pi~~tl~f~r~~z was absent on Day 0. but was isolated in increasing frequencies on subsequent sampling days. Chr~wsporium pannarunl was isolated from control soils and those treated with Captan, Dicloran and Thirsm only in low frequencies and in the colder months. However, in Verdasan-treated soil. C. pun-
P = 0.05; i indicates
an increase
norutn was isolated throughout the year with a general abundance in winter and with higher frequencies on Day 0 than on other sampling days. C~~ld~sporiu~ ~lado.s~~riaid~~~ was isolated from control soil mainly in the summer and autumn months when it contributed significantly to the general abundance of fungal isolates at that time. None of the four fungicides significantly altered either its numbers or its occurrence, although on several Day 0 samples of Verdasan-treated soil, it occurred in reduced frequencies.
Fungicides
from control
Table 3. The mean number of species isolated and fungicide-treated soils
Control Captan Dicloran Thiram
and soil fungi
Day 0
Day 7, 14 and 28
I1
II 10
(t = Y37, 10
10
(C = ::92,
(t = “jB,
(f =%5f
(E = Yl9,
Verdasan
* Significantly parenthesis.
different
from
control;
t values
given in
G~~ociudiumroseum, Gliomastis mliroru~n (Corda) Hughes var. felina (Marchal~ Hughes, ~~}njcol~~usc~ atra and ~epedoniuff7 c~~r~.~ospernzu~n were all either eliminated or isolated only in reduced frequencies from Day 0 samples of fungicide-treated soils. Their numbers recovered rapidly on subsequent sampling days. ~ort~ere~la minut~sima showed a summer abundance in control soils and was not recorded in the winter and spring months. During this natural seasonal incidence of the species, it was isolated from soils treated with all four fungicides both on Day 0 and on subsequent sampling days. Trichocladium asperum showed a winter and spring abundance in control soil. Its occurrence in Diclorantreated soil was generally similar to that in control Table 4. The fungal
numbers
Mean number of propagules g- ’ soil Unstored Stored Control 0 I 14 28 Captan 0 7 14 28 Dicloran 0 7 14 28 Thiram 0 1 14 28 Verdasan 0 9 14 28
301
soil. In Captan, Thiram and Verdasan-treated soils, it was either eliminated or isolated only in reduced numbers in the summer months. In the other months, it was isoiated both on Day 0 and on subsequent sampling days from all fungicide-treated soifs. Trichoderma humatum was isolated throughout the year from both control and treated soils on Day 0 and on the other sampling days. Trichoderma uiride (sensu Rifai, 1969) was eliminated from Day 0 samples treated with Captan, Thiram and Verdasan although in control soil it was isolated throughout the year. Its numbers built up rapidly on Day 7, 14 and 28 when it was often isolated at lOO?< frequency. In Dicloran-treated soils. 7: uiride was not eliminated although its numbers were reduced following the application of the fungicide. Z~gar~~~nc~l{smoeki was consistently isolated from both control and treated soils on Day 0 and on subsequent sampling days. As with Trichader~)~a hamatum, the occurrence and numbers of Z. moelieri were not altered by any of the four fungicides. In general, the species composition of Diclorantreated soil was similar to that of control soil. The species composition of soils treated with the other three fungicides were different on Day 0 than on the other sampling days. In Captan and Thiram-treated soils, Penicillium spp were amongst the most frequently isolated fungi throughout the year. While P. pulvillorum was isolated in increasing frequencies on days 7, 14 and 28 from Thiram-treated soil, other Pen~ci~~~~rnspp were isolated only infrequently from Verdasan-treated soil. in stored
and unstored
Mean number of isolations per 5 replicate soil plates Unstored Stored
soil
Mean
number of species Stored Unstored
19.6* 11.3* 16.4* 15.0*
25.4 27.1 27.5 26.9
37 36 3Y‘ 34*
42 40 41 42
9 10 10 9
11 10 10 10
22.P 23.9 23.5 22.3
19.4 23.8 25.6 26.9
34t 36 40 35
29 34 38 39
10 9 11 10
9 10 11 11
20.8 19.7’ 19.6* 17.8*
22.6 25.1 2-l. 1 26.7
35 39 41 41
33 38 42 40
8 11 II 10
10 IO 11 10
15.5 16.5 16.7* 16.6*
16.2 18.3 20.5 21.1
30t 37t 39t 48t
25 30 33 37
9 I1 13t 13
8 9 10 11
16.4? 14.3 14.9* 15.5*
12.7 15.6 18.5 19.2
20 26 33 37t
19 24 29 32
5 7 9 11
5 7 9 10
* Significantly lower than stored soil P = 0.05.
value in unstored
soil P = 0.05; t: significantly
higher
than
value
in un-
302
A.J.
KUTHUBIJTHEEN
In order to determine whether 7’richoderma uiride arose from new inocula recolonizing from non-fungicide treated parts of the soil or if it was from inoculum that had survived the fungitoxic effects of the fungicides, germination experiments were carried out (Kuthubutheen and Pugh, 1978). SoiI extract agar containing such fungicide concentrations as would inhibit the germination of spores of 7: uiride, was inoculated with spores and subsequently leached by washing in sterile distilled water. All concentrations used of Captan and Dicloranwere found to be only fungistatic. The spores of 7: vi&e germinated in the washed agar and the fungi growing in soils following treatment could therefore have survived the treatment or may have reinvaded from surrounding soils. However, with Thiram and Verdasan even low initial concentrations were fungicidal. The effects of storage
Stored soil samples generally differed from the unstored soils only in the number of propagules g- ’ soil or in the number of isolations but not in the actual number of species obtained from them (Table 4). The changes in fungai numbers in the stored soils were therefore either due to a proliferation or to poor sporulation of those fungi that survived the effects of the fungicides. The mean number of propagules in the Day 0 samples of Captan-treated soil, when stored, increased to be similar to that in unstored control soils. This could indicate that Captan is readily degraded biologically and those fungi that survived could proliferate and increase in numbers when the fungitoxicity of Captan is lost. The increase in the numbers of propagules in the stored Day 0 samples of Verdasan-treated soils was due largely to C. pannorum. B. pilul~~erum, G. roseum, Penicillium spp. and T. viride and Z. moelleri were the most frequently isolated fungi from fungicide-treaty stored soils. DlSCUSSlON
Although fungicides are not always directly applied to soil, large quantities of these chemicals reach the soil from aerial parts of treated plants. Because many of these compounds are not pathogen specific, a wide variety of non-pathogenic saprophytic fungi are also eliminated. Pugh and Williams (1971) found that the fungal numbers in the golf-greens that had received about 20yr treatment of Verdasan were significantly reduced compared with those in the untreated fairways. They found that the dilution plate and soil plate counts in the treated soils were reduced by 97% and 64”/;1 respectively compared with the control soil. Comparable figures after 1 year of treatment were 38% and 37x, although immediately after spraying, the reduction was 50% and 55% respectively. From the two studies it is evident that the longterm effects of Verdasan on soil fungal populations are cumulative. The toxicity of Verdasan is enhanced by its persistence and by its breakdown products. Kimura and Miller (1964) found that a large portion of the organic mercurial compounds applied to soil was found to be in the organo-mercury form after a lapse of 3&50 days. Of the phenylmercury acetate applied, 6t&70°A was extractable as the intact phenylmercury compound. Degradation of PMA and other
and G. J. F.
PUGH
organo-mercury compounds produced metallic mercury. .I. I. Williams (1973. unpublished Ph.D. Thesis, Umversity of Nottingham) showed that the total mercury in the golf greens was 7.62 pg. g- ’ soil compared soil in the untreated fairway. with 0.61~g.g-i Soil fungal population was reduced more by Verdasan and Thiram than by Captan and Dicloran. Similarly, Wainwright and Pugh (1973) showed that nitrification in soil was inhibited more by Verdasan than by Thiram, Captan and Dicloran in that order. Although Thiram and Verdasan caused the most deleterious effects on soil fungal numbers. cetlulolytic species such as A. verruculosa, Ckuetomium spp., G. roseum, Penicillium spp. and Trichoderma spp. were not completely eliminated by these fungicides. However, the principal species, in Verdasan-treated soils was C. pannorum which was found to be a slow growing and only moderately cellulolytic fungus (Kuthubutheen, unpublished data). The abundance of C. punnorurn in Verdasan-treated soil may be attributed to the elimination of its faster-growing competitors by the fungicide. However, its abundance in Verdasantreated soil and not in Thiram-treated soil. points more towards the fact that the fungus can detoxify and utihse Verdasan rather than to its proliferation in the absence of faster-growing competitors. Wilhams and Pugh (1975) showed that C. punnorum was capable of detoxifying the fungicide and utilizing it as a carbon source. Corden and Young (1965) found that the application of MetasoI (methy~mercury-S-hydroxyquinolate) resulted in the rapid reduction in the fungal population. The subsequent increase in the recolonizing population was primarily due to PenicilIium spp. and a Chrysosporium sp., given as Sporotrichum (Carmichael, 1962). C. partnoruln was also reported by Poole and Price (1971) as occurring in increased numbers in soils receiving incremental cellulose. However, it is highly probable that mercury in the effluent of paper mills situated in the vicinity of their site of study could have caused the prolific numbers of C. pannorum (Williams and Pugh, 1975). While C. pannorum occurred in increased frequencies in Verdasan-treated soil on Day 0, 7: ciride and B. pi~u~~orunl were sensitive to Verdasan and were eliminated on Day 0. The relative abundance of these species can be used to indicate the levels of pollution of the soil by organo-mercurial fungicides. However, C. pannorum also occurs abundantly in cold soils (Ivarson, 1973) because of its psychrophilic abilities. It is important that these two situations are differentiated. The marked reductions in fungal numbers caused by Thiram could be attributed to its breakdown products which are in themselves potent fungitoxic substances. In soil, Thiram is reduced to dimethyldithiocarbomate and finally to carbon disulphide and dimethylamine. Conversely, the rapid recolonization of Captan and Dicloran-treated soils could be attributed to the shorter persistence of these fungicides. Although Lukens and Sisler (1958) proposed that Captan is degraded by cellular thiols in Go into a toxic compound thiophosgene, Captan has been shown to have extremely low persistence (Burchfield, 1960; Griffiths and Mathews, 1969: Agnihotri. 1971).
Fungicides and soil fungi Species of Trichoderma have been consistently encountered as major recolonizers of fungicide- and fumigant-treated soils (e.g. Bliss, 1951; Evans, 1955; Saksena 1960; Moubasher 1963. Danielson and Davy. 1969; Agnihotri, 1973, 1974; Peeples, 1974; Catska and Vrany, 1976). Many of these studies were published before Rifai’s (1969) revision of the genus Trichoderma. Mughogho (1968) analysed the species composition of Trichoderma found in treated soils with the aid of the Rifai (1969) key and showed that several species including 7: ~~~~~~~z~~, 7: ~zar~iunum. 7: k~~?~in~~ and 7: riride were consistently present. Few studies have provided evidence to prove that 7: r!iride is a recolonizer rather than a survivor that invades and proliferates on removal of the toxicant. While acknowledging the problems of extrapolating from the laboratory to field conditions, the growth of T. r:iride sense Rifai in soils following the application of Thiram and Verdasan was most probably due to recolonization from outside the treated area. When T. hamatum SV~SURifai was similarly treated, it was found to be more tolerant of the four fungicides used. Growth of this species after treatment of soil with organo-mercurials. therefore. could be from surviving inocula and from recolonization. There is therefore a need to bear in mind the different behavioural patterns of growth of the species Trichoderrna which were previously grouped together as 7: viride by Bisby (1939) but separated by Rifai (1969). Future workers must separate the species of this genus to help gain a fuller understanding of their contribution to soil processes.
M. E. and YOUNG R. A. (1965) Changes in the soil microflora following fungicide treatment. Soil Sci. 99, 272-277. DANIELSON R. M. and DAVE%’C, B. (1969) Microbiat recolonization of a fumigated nursery soil. For. Sci. 15, 368-380. DOMSCH K. H. (1964) Soil fungicides. A. Rrr. Phytopath. 2, 293-320. DOMSCH K. H. (1970) Effects of fungicides on microbial nooulations in soil. In Pesticides in fhe Soil: Ecoloyy. b&&rion and Mowment. Int. Symp. on Pesticides in the Soil, pp. 42--46. Michigan State University. E. Lansing. &C;INS H. 0. W. and PUGH G. J. F. (1962) isolation of cellulose decomposing fungi from soil. Natlrre 1% 94-95. EVANS E. (1955) Survival and recolonization by fungi in soil treated with formalin or carbon disulphide. Trans. Br. myof. SW. 38, 335-346. FISHER R. A. and YATES F. (1963) Sr~~~i,~rjr~~Tuhks fir Ej~~~gjc~~, Agricuhrui and Metlicui Resecrrch. Oliver & Boyd. London. GRIFFITHS R. L. and MATHEWS S. (1969) The persistence in soil of the fungicidal seed dressings captan and thiram. Ann. appl. Biol. 64, 113-118. &ARSON K. C. (1973) Fungal flora and rate of decomposition or leaf litter at low temperatures. Cun. J. Soil Sci. 53, 79-84. KIMURA Y. and MILLER V. L. (1964) The degradation of organomercury fungicides in soil. J. Agric. Frf Chrm. 12, 253.-257. KREUTZER W. A. (1965) The reinfestation of treated soil. In Eco/c?y~ of Soil-hornc Plunt Parhoyrns (K. F. Baker and W. C. Synder, Eds), pp. 495-507. IJniv. California Press. KUTHUBK~THE~NA. J. and PUCH G. J. F. (1978) Effects of fungicides on the physiology of phylloplane fungi.
CORDEN
Trans. Br. my&.
ilck~~o~l~‘d~~tt~e,lt.s -.We thank the University of Malaya for a Tutorship Grant to KAJ; the University of Nottingham for the use of the grassland site; Plant Protection Ltd. for supplying the fungicides and Mr P. Smithurst for his technical assistance.
REFERENCES
AGNIHOTRI V. P. (1971) Persistence of C’aptan and its effects on microflora, respiration and nitrification of a forest nursery soil. Can. J. ~~~~~~bj~)~.17, 377-383. AC;NIHOTRI V. P. (1973) Effects of dexon on soil microflora and their ammonification and nitritication activities. Iu~. J. r.Xp. Biol. 11, 213 216. AGNIHOTRI V. P. (1974) Thiram induced changes in the soil microflora, their physiological activity and control of damping-off in chillies (Capsicrm mntm). fntf. J. mp. Biol. 12, 85-88. BISBY G. R. (1939) ~rj~~o~~~~~~~ z+ride Pers. ex Fr. and notes on H~p~c’rer~. Trans. Br. myrol. Sot. 23, 149%168. BLISS E. D. (1951) The destruction of Armilfaria mefiea in citrus soils. Phytopothology 41, 665-683. BUKCHFIEL~ H. P. (1960) Performance of fungicides on plants and in soil---physical. chemical and biological considerations. In Plarzf Pathology; an Atitmced Treurise. (J. H. Horsfall and A. E. Dimond, Eds). Vol. 3. pp. 477 520. Academic Press, New York. CARMI~HAEI J. W. (1962) Ci~r~~~porirr!n and some other aleuriosporic hypomycetes. CU~Z.J. Bar. 40, I 137- 1173. CATSKA V. and VRAXY J. (1976) Rhizosphere mycoflora of wheat after foliar application of chlorocholine chloride, urea and 4-chloro-2-mcthylhenoxyacetic acid. Folia ~~icn,hio!. 21, 268--27X
303
Sot. 71, 261-269.
LUKENS R. J. and SIXER H. D. (1958) Chemical reactions involved in the fungitoxicity of captan. Ph~topatholoq~ 48, 235-244. MOUBASH~R A. H. (1963). Selective effects of fumigation with carbon disillphide on soil fungus flora. Trrrtzs. Br. i,Z~CCli.sot. 46, 338--344. MUGHO~;H~ L. K. (1968) The fungus flora of fumigated soils. Trans. Br. J)I~CO/. Sue. 51, &l--459. PEFPLES J. L. (19741 Microbial activity in benomvl-treated soils. Phyro&doyy 64, 857 860. . Poo~t N. J. and PRICC P. C. (1971) The occurrence of C~r~.sff.~~~rjz{~ ~~~,*~~~~ in soils receiving incremental celtulose. Soit &of. Bj~~~~z~~,3, 161-166. PUGH G. J. F. and WILLIAMS J. 1. (1971) Effects of an organo-mercury fungicide on saprophytic fungi and on litter decomposition. Trans. Br. ~lyco/. Sot. 57, I64 166. RIFAI M. A. (1969) A revision of the genus Trichodermu. Mycol. Papers 116, 9-56. SAKSENA S. B. (1960) Effects of carbon disulphide fumigation on ~~jc~z~~~~~~f?~u riri& and other soil fungi. Tr~ns. Br. rrr)coi. sot. 43, 1 1 I-1 16. WAINWRIGHK M. (1977) Effects of fungicides on the microbiology and biochemistry of soils. 2. F@nzenernaehr. RodenKd. 140, 587-603. WAINWRIC~HTM. and PUGH G. J. F. (1973) The effect of three fungicides on nitrification and ammonification in soil. Soil Biol. Biochrm. 5, 577-584. WAIN~RI~HT M. and PUGH G. J. F. (1975) Effect of rungitides on the numbers of micro-organisms and frequency of cellulolytic fungi in soils. PI. Soii 43, 561 572. WARCUP J. H. (1950) The soil-plate method for isolation of fungi from soil. Nature, Land. 166, 117. WIL.LIAMS J. I. and PLJC;H G. J. F. (1975) Resistance of Chrysosporium p~nnorum to an organo-mercury fungicide. Truths. Br. m_~co/. Sot. 64. 255.-256.
B,ochum. Vol 1I. pp. 291to303 0 PergamonPress Ltd 1979. Pnnted m Great Br~tam
0038.0717/79'0601-029750200/0
THE EFFECTS OF FUNGICIDES FUNGAL POPULATIONS A. J. Department
of Biological
and G.J.
KUTHUBUTHEEN*
Sciences,
University
(Accrprrci
of Aston,
3 NoNmhrr
F.
Gosta
ON SOIL
PUGH
Green,
Birmingham,
B4 7ET.
197X)
Summary-Four fungicides, Captan. Dicloran, Thiram and Verdasan were applied at 2%day intervals for 12 consecutive months and their effects on soil fungal numbers and the incidence of individual species were studied. Immediately after application, these fungicides reduced the number of fungal compared with control. Captan- and Dicloranpropagules in soil by 23, I I, 36 and 50% respectively treated soils were rapidly recolonised within 7 days of the application of fungicides. The effects of Thiram and Verdasan were more persistent: the fungal numbers in soils treated with these fungicides did not recover sufficiently to reach control levels throughout the sampling period. Fewer species of fungi were isolated from Thiram- and Verdasan-treated soils than from Captan- and Dicloran-treated soils. Chrysosporium pannorum (Link) Hughes was the principal species isolated from Verdasan-treated
soil and the fungus was isolated in increased numbers immediately after application of Verdasan than on subsequent sampling days. Cladosporium cladosporioides (Fres.) de Vries, Morrierella minurissima van Tiegh, Trichocladium asperum Harz, Trichoderma humarum (Bon.) Bain. and Zyyorhynchus moelleri Vuill. were found to be generally tolerant of all four fungicides. However, Bofryotrichum piluliferum Sacc. and Marchal, Gliocladium roseurn Bain., Humicola @co-atra Traaen, Sepedonium chrysospermum (Bull.) Link ex Fr., and Trichodermn oiride Pers. ex Fr. were generally intolerant of the fungicides but rapidly recolonised the treated soils. While the concentrations of Captan and Dicloran used were fungistatic to 7: ciride, Thiram and Verdasan were fungicidal.
MATERIALS
INTRODUCTION
The changes in the fungal population of soils treated with fungicides may be due to the direct effect of the chemicals on soil fungi. Because most cellular processes are common to all fungi, fungicides kill or adversely affect both the target fungi and a wide range of non-target species (Domsch, 1964, 1970; Kreutzer, 1965; Wainwright, 1977). The changes in the fungal population of treated soils may also, however, be due to the alteration of the physico-chemical characteristics of the soil by fungicides. The application of the organo-mercurial fungicide Verdasan, for example, was shown to reduce decomposition and contribute to an accumulation of organic matter (Pugh and Williams, 1971). This in turn could result in waterlogging and anaerobic conditions which would further reduce microbial activity. Pugh and Williams (1971) studied the fungal populations of a golf-course in Nottingham to which Verdasan had been added twice a year since 1953. Wainwright and Pugh (1975) followed the changes in fungal populations after a single application of some commonly used fungicides. Our aim was to explore the changes brought about by successive monthly applications of fungicides and in doing so, to link the earlier studies. The long-term effects of the fungicides on the soil mycoflora were monitored to determine successional patterns and to distinguish between tolerant and recolonising species.
* Present address: Department of Botany. Malaya, Kuala Lumpur 22-11, Malaysia. S.RB,1,3--P
University
of
AND
METHODS
An 8 x 8 m latin square plot was laid out on a grassland site at the University of Nottingham. To facilitate spraying and to prevent cross-contamination, the plot was divided into 25 sub-squares (1 x 1 m), each bounded by a path 0.5 m. The soil was a sandy-loam with a mean pH 6.8; water holding capacity, 34.7 ml lOOg_-’ soil; loss of weight on ignition, 2.72”;; and total N, 0.169/,. Starting from 9 February 1975. the plots were sprayed at 28-day intervals with the following fungicides: Captan- a.i. SOY/;;(w/w) N-trichloromethylthio-4-cyclohexene-1.2,dicarboximide; Dicloran-a.i. 4”,‘, (w/w) 2,6-dichloro-4-nitroaniline; Thiram-a.i. 501;, (w/w) tetramethylthiuram disulphide and Verdasana.i. 2.5”~; (w/w) organically combined mercury. Each fungicide treatment occurred once in each row and once in each column of the plot. The five untreated squares served as control and were sprayed with tapwater. The fungicides were applied at the following rates: Captan 9 kg ha-‘; Dicloran 2 kg ha-‘; Thiram 6.7 kg ha- ’ ; and Verdasan 12.5 kg ha- I. Appropriate amounts of the fungicides were mixed in the same volume of water. Using random sampling numbers (Fisher and Yates, 1963). soil cores (2-3 cm deep) were taken immediately after spraying and subsequently after 7, 14 and 28 days. The samples taken immediately after spraying were designated Day 0 samples. A total of 48 such samples (including 12 immediately after spraying) were taken from both the control and treated plots. The soil samples from the 5 replicate plots of each treatment were mixed and the fungal 297
A. J.
298
KUTHURUTHEEN and G. .I F’. PN;H
numbers were assessed within 24 h of collection. The seven soil samples from Day 2X of the November 1975 cycle to Day 0 of the January 1976 cycle, however. were stored at 4,C and plated out subsequently. The dilution method was used to assess the gross number of fungal propagules present in soil while the soil plate method (Warcup, 1950) was used to determine the frequency and occurrence of species. Cellulose agar (Eggins and Pugh, 1962) was used for both methods. The presence of a species on one of the five replicate soil plates was considered as one isolation with a frequency of 2(X,,. The plates were incubated at 25 C and examined after 7 days and subsequently at regular intervals until no new colonies or species were observed. RESULTS
The changes in the number of propagules in soils treated with the fungicides are shown in Figs la-b. In the control plots, lowest numbers of fungi were obtained from about December to March while there was a general abundance in the other months. This pattern of occurrence was not altered in Captan- and Dicloran-treated soils. The application of Thiram and Verdasan. however. suppressed the peaks of occurrence of fungi in the treated soils. The fungal counts obtained with Day 0 samples of the fungicide-treated soils were generally lower than those obtained with control and Day 7, 14 and 28 samples. The mean numbers of prop~lgules in the Day 0 samples of C’aptan-. Thiram. and Verdasan-treated soils from February to October were significantly lower compared with control. (Fig. 2). On Days 7. 14 and 2X. however, the fungal numbers in the Cap-
Fig. 1b. The effects of Dicloran and Thiram on the mean number of hrngal propagules in soil from FcbOct, B Thlram: l Control: 0 0 Dicforan: 0 0 7 funglcidc application.
tan-treated soil increased to be comparable with that of control. The etfccts of Thiran and Verdasan were more persistent; the fungal numhcrs in soils trcatcd with these fungicides did not recover sufficiently to be comparable with control numhcrs. The mean fungal numbers in Dicloran-treated soil on all four sampling periods were not significantly lower than that of the control soil. In general. the fungal numbers were reduced most by Verdasan, then by Thiram. Captan and Dicloran in that order. The reductions in the mean number of propagules after application of Captan. & -’ soil immediately Thiram and Verdasan were significant compared with control (Table 1). Mowever. in the Day 7. 14 and 2X samples, the reductions in mean numbers of propagules from only the Thiram- and Verdasan-treated soils remained significant. Soil plute method
Fig. la. The etl’ects of Captan and Verdasan on the mean of fungal propagules in soil from Feb-Oct. Control: A----- a Captan; 0-n Verdasan;
number 0 -*
1 fungicide
application.
The results obtained with the soil plate method were comparable with those observed with the dilution method. In the control soils, lower number of isolations were obtained from about December to March white there was a general abundance in the other months. While these periods of peak isolations were evident in the Captan- and Dicloran-treated soils, in the Thiram- and Verdasan-treated soils the periods of general abundance were absent. The mean number of isolations obtained from Thiram- and Verdasan-treated soils throughout the study did not recover suthciently to be comparable with control (Fig. 3). The mean number of isolations from Captan- and Dicloran-treated soils were significantly lower than control on Day 0. Subsequently, the soils treated with these fungicides were rapidly recolonised so that the fungal numbers were comparable with control.
299
Fungicides and soil fungi
l-
>-
b
0
14
I
Sampling
20
period (days)
Fig. 2. The mean number of propagules on Day 0, 7, 14 and 28 in control and treated soils from Thiram; 0-O Verdasan. FebOct. O---O Control; A---A Captan; o--<) Dicloran; (Vertical bars show 95% confidence limits.)
The reductions in the mean number of isolations immediately after application of all four fungicides were significant compared with control (Table 2). However in the Day 7, 14. and 28 samples, the reductions in the mean number of isolations from only the Thiram- and Verdasan-treated soils remained significant. The periods of peak occurrence for the number of propagules and for the number of isolations in the control soil corresponded to the period when the maximum number of species were isolated from the soil plates. Generally, immediately after application of the fungicides, fewer species of fungi were isolated from the fungicide-treated soils than from control soils. The effects of Thiram and Verdasan were more deleterious; only 5-7 species were consistently isolated immediately after application of the fungicides (Table 3). The mean number of isolations and species were reduced most by Verdasan, then by Thiram, Captan and Dicloran, in that order. Cladosporium cladosporioides, Mortierella minutissima, Trichocladium asperum, Trichoderma hamatum and Zygorhynchus moelleri were found to be generally
tolerant of all four fungicides. These fungi were isolated in comparable frequencies both immediately
after spraying and on subsequent sampling days. However, Botryotrichum piluliferum, Gliocladium roseum, Humicola fisco-atra, Sepedonium chrysospermum and Trichoderma viride were generally intolerant
of the fungicides. These fungi were either absent or were isolated only in low frequencies immediately after application. But subsequently the fungi rapidly recolonized the treated soils and were therefore identified as the recolonizer species. The principal recolonizing fungi of Thiram-treated soils were Penicillium spp. and 7: viride. Of the species isolated, Apiosordaria uerruculosa (Jensen) von Arx and Gams, was isolated throughout the year in control soils, with a minimum occurrence between August and September. It was tolerant of Captan, Dicloran and Thiram and was isolated on Day 0 from soils treated with these fungicides. Its numbers tended to increase on subsequent sampling days. A. verruculosa, however, was not isolated on any of the Day 0 samples of Verdasan-treated soil. It occurred in Verdasan-treated soils only in the winter months on Days 7, 14 and 28. Botryotrichum piluliferum was isolated throughout the year in control soils with a general abundance in the summer and autumn months. In Day 0 samples
A, J.
300
KUTHUBUTHEEN and
Sampling Fig. 3. The mean number Feb-Oct. O-,--• Control;
Table
1. Reduction
$0 (days)
in the mean number of propagules g-’ percentage of control
2. Reduction
soil as a
Day 0
7
14
28
23* II 36* 50*
12 5 32* 42*
7 I 26* 33*
(t0.3) 0.8 22* 29s
* Significantly different compared with control.
Captan Dicloran Thiram Verdasan
period
of isolations on Day 0. 7, 14 and 28 from 45 replicate soil plates from Dicloran; 0-0 Thiram; U----U Verdasan. A--A Captan; (Vertical bars show 957; confidence limits.)
Captan Dicloran Thiram Verdasan
Table
PUGH
A
i
i
G. J. F.
from control
P = 0.05; _t indicates
in the mean number of control
of isolations
an increase
as a percentage
Day 0
7
14
28
31* 21’ 41* 55*
14* 6 25* 41*
c+:.5, 20* 31*
7 3 12* 23*
* Si~i~cantiy different compared with control.
from control
of Captan-treated soil, B. pilulifirurn was either ehminated or isolated only at reduced frequencies. While its numbers were not markedly reduced in Diclorantreated soil, in Thiram and Verdasan-treated soils, I?. pi~~tl~f~r~~z was absent on Day 0. but was isolated in increasing frequencies on subsequent sampling days. Chr~wsporium pannarunl was isolated from control soils and those treated with Captan, Dicloran and Thirsm only in low frequencies and in the colder months. However, in Verdasan-treated soil. C. pun-
P = 0.05; i indicates
an increase
norutn was isolated throughout the year with a general abundance in winter and with higher frequencies on Day 0 than on other sampling days. C~~ld~sporiu~ ~lado.s~~riaid~~~ was isolated from control soil mainly in the summer and autumn months when it contributed significantly to the general abundance of fungal isolates at that time. None of the four fungicides significantly altered either its numbers or its occurrence, although on several Day 0 samples of Verdasan-treated soil, it occurred in reduced frequencies.
Fungicides
from control
Table 3. The mean number of species isolated and fungicide-treated soils
Control Captan Dicloran Thiram
and soil fungi
Day 0
Day 7, 14 and 28
I1
II 10
(t = Y37, 10
10
(C = ::92,
(t = “jB,
(f =%5f
(E = Yl9,
Verdasan
* Significantly parenthesis.
different
from
control;
t values
given in
G~~ociudiumroseum, Gliomastis mliroru~n (Corda) Hughes var. felina (Marchal~ Hughes, ~~}njcol~~usc~ atra and ~epedoniuff7 c~~r~.~ospernzu~n were all either eliminated or isolated only in reduced frequencies from Day 0 samples of fungicide-treated soils. Their numbers recovered rapidly on subsequent sampling days. ~ort~ere~la minut~sima showed a summer abundance in control soils and was not recorded in the winter and spring months. During this natural seasonal incidence of the species, it was isolated from soils treated with all four fungicides both on Day 0 and on subsequent sampling days. Trichocladium asperum showed a winter and spring abundance in control soil. Its occurrence in Diclorantreated soil was generally similar to that in control Table 4. The fungal
numbers
Mean number of propagules g- ’ soil Unstored Stored Control 0 I 14 28 Captan 0 7 14 28 Dicloran 0 7 14 28 Thiram 0 1 14 28 Verdasan 0 9 14 28
301
soil. In Captan, Thiram and Verdasan-treated soils, it was either eliminated or isolated only in reduced numbers in the summer months. In the other months, it was isoiated both on Day 0 and on subsequent sampling days from all fungicide-treated soifs. Trichoderma humatum was isolated throughout the year from both control and treated soils on Day 0 and on the other sampling days. Trichoderma uiride (sensu Rifai, 1969) was eliminated from Day 0 samples treated with Captan, Thiram and Verdasan although in control soil it was isolated throughout the year. Its numbers built up rapidly on Day 7, 14 and 28 when it was often isolated at lOO?< frequency. In Dicloran-treated soils. 7: uiride was not eliminated although its numbers were reduced following the application of the fungicide. Z~gar~~~nc~l{smoeki was consistently isolated from both control and treated soils on Day 0 and on subsequent sampling days. As with Trichader~)~a hamatum, the occurrence and numbers of Z. moelieri were not altered by any of the four fungicides. In general, the species composition of Diclorantreated soil was similar to that of control soil. The species composition of soils treated with the other three fungicides were different on Day 0 than on the other sampling days. In Captan and Thiram-treated soils, Penicillium spp were amongst the most frequently isolated fungi throughout the year. While P. pulvillorum was isolated in increasing frequencies on days 7, 14 and 28 from Thiram-treated soil, other Pen~ci~~~~rnspp were isolated only infrequently from Verdasan-treated soil. in stored
and unstored
Mean number of isolations per 5 replicate soil plates Unstored Stored
soil
Mean
number of species Stored Unstored
19.6* 11.3* 16.4* 15.0*
25.4 27.1 27.5 26.9
37 36 3Y‘ 34*
42 40 41 42
9 10 10 9
11 10 10 10
22.P 23.9 23.5 22.3
19.4 23.8 25.6 26.9
34t 36 40 35
29 34 38 39
10 9 11 10
9 10 11 11
20.8 19.7’ 19.6* 17.8*
22.6 25.1 2-l. 1 26.7
35 39 41 41
33 38 42 40
8 11 II 10
10 IO 11 10
15.5 16.5 16.7* 16.6*
16.2 18.3 20.5 21.1
30t 37t 39t 48t
25 30 33 37
9 I1 13t 13
8 9 10 11
16.4? 14.3 14.9* 15.5*
12.7 15.6 18.5 19.2
20 26 33 37t
19 24 29 32
5 7 9 11
5 7 9 10
* Significantly lower than stored soil P = 0.05.
value in unstored
soil P = 0.05; t: significantly
higher
than
value
in un-
302
A.J.
KUTHUBIJTHEEN
In order to determine whether 7’richoderma uiride arose from new inocula recolonizing from non-fungicide treated parts of the soil or if it was from inoculum that had survived the fungitoxic effects of the fungicides, germination experiments were carried out (Kuthubutheen and Pugh, 1978). SoiI extract agar containing such fungicide concentrations as would inhibit the germination of spores of 7: uiride, was inoculated with spores and subsequently leached by washing in sterile distilled water. All concentrations used of Captan and Dicloranwere found to be only fungistatic. The spores of 7: vi&e germinated in the washed agar and the fungi growing in soils following treatment could therefore have survived the treatment or may have reinvaded from surrounding soils. However, with Thiram and Verdasan even low initial concentrations were fungicidal. The effects of storage
Stored soil samples generally differed from the unstored soils only in the number of propagules g- ’ soil or in the number of isolations but not in the actual number of species obtained from them (Table 4). The changes in fungai numbers in the stored soils were therefore either due to a proliferation or to poor sporulation of those fungi that survived the effects of the fungicides. The mean number of propagules in the Day 0 samples of Captan-treated soil, when stored, increased to be similar to that in unstored control soils. This could indicate that Captan is readily degraded biologically and those fungi that survived could proliferate and increase in numbers when the fungitoxicity of Captan is lost. The increase in the numbers of propagules in the stored Day 0 samples of Verdasan-treated soils was due largely to C. pannorum. B. pilul~~erum, G. roseum, Penicillium spp. and T. viride and Z. moelleri were the most frequently isolated fungi from fungicide-treaty stored soils. DlSCUSSlON
Although fungicides are not always directly applied to soil, large quantities of these chemicals reach the soil from aerial parts of treated plants. Because many of these compounds are not pathogen specific, a wide variety of non-pathogenic saprophytic fungi are also eliminated. Pugh and Williams (1971) found that the fungal numbers in the golf-greens that had received about 20yr treatment of Verdasan were significantly reduced compared with those in the untreated fairways. They found that the dilution plate and soil plate counts in the treated soils were reduced by 97% and 64”/;1 respectively compared with the control soil. Comparable figures after 1 year of treatment were 38% and 37x, although immediately after spraying, the reduction was 50% and 55% respectively. From the two studies it is evident that the longterm effects of Verdasan on soil fungal populations are cumulative. The toxicity of Verdasan is enhanced by its persistence and by its breakdown products. Kimura and Miller (1964) found that a large portion of the organic mercurial compounds applied to soil was found to be in the organo-mercury form after a lapse of 3&50 days. Of the phenylmercury acetate applied, 6t&70°A was extractable as the intact phenylmercury compound. Degradation of PMA and other
and G. J. F.
PUGH
organo-mercury compounds produced metallic mercury. .I. I. Williams (1973. unpublished Ph.D. Thesis, Umversity of Nottingham) showed that the total mercury in the golf greens was 7.62 pg. g- ’ soil compared soil in the untreated fairway. with 0.61~g.g-i Soil fungal population was reduced more by Verdasan and Thiram than by Captan and Dicloran. Similarly, Wainwright and Pugh (1973) showed that nitrification in soil was inhibited more by Verdasan than by Thiram, Captan and Dicloran in that order. Although Thiram and Verdasan caused the most deleterious effects on soil fungal numbers. cetlulolytic species such as A. verruculosa, Ckuetomium spp., G. roseum, Penicillium spp. and Trichoderma spp. were not completely eliminated by these fungicides. However, the principal species, in Verdasan-treated soils was C. pannorum which was found to be a slow growing and only moderately cellulolytic fungus (Kuthubutheen, unpublished data). The abundance of C. punnorurn in Verdasan-treated soil may be attributed to the elimination of its faster-growing competitors by the fungicide. However, its abundance in Verdasantreated soil and not in Thiram-treated soil. points more towards the fact that the fungus can detoxify and utihse Verdasan rather than to its proliferation in the absence of faster-growing competitors. Wilhams and Pugh (1975) showed that C. punnorum was capable of detoxifying the fungicide and utilizing it as a carbon source. Corden and Young (1965) found that the application of MetasoI (methy~mercury-S-hydroxyquinolate) resulted in the rapid reduction in the fungal population. The subsequent increase in the recolonizing population was primarily due to PenicilIium spp. and a Chrysosporium sp., given as Sporotrichum (Carmichael, 1962). C. partnoruln was also reported by Poole and Price (1971) as occurring in increased numbers in soils receiving incremental cellulose. However, it is highly probable that mercury in the effluent of paper mills situated in the vicinity of their site of study could have caused the prolific numbers of C. pannorum (Williams and Pugh, 1975). While C. pannorum occurred in increased frequencies in Verdasan-treated soil on Day 0, 7: ciride and B. pi~u~~orunl were sensitive to Verdasan and were eliminated on Day 0. The relative abundance of these species can be used to indicate the levels of pollution of the soil by organo-mercurial fungicides. However, C. pannorum also occurs abundantly in cold soils (Ivarson, 1973) because of its psychrophilic abilities. It is important that these two situations are differentiated. The marked reductions in fungal numbers caused by Thiram could be attributed to its breakdown products which are in themselves potent fungitoxic substances. In soil, Thiram is reduced to dimethyldithiocarbomate and finally to carbon disulphide and dimethylamine. Conversely, the rapid recolonization of Captan and Dicloran-treated soils could be attributed to the shorter persistence of these fungicides. Although Lukens and Sisler (1958) proposed that Captan is degraded by cellular thiols in Go into a toxic compound thiophosgene, Captan has been shown to have extremely low persistence (Burchfield, 1960; Griffiths and Mathews, 1969: Agnihotri. 1971).
Fungicides and soil fungi Species of Trichoderma have been consistently encountered as major recolonizers of fungicide- and fumigant-treated soils (e.g. Bliss, 1951; Evans, 1955; Saksena 1960; Moubasher 1963. Danielson and Davy. 1969; Agnihotri, 1973, 1974; Peeples, 1974; Catska and Vrany, 1976). Many of these studies were published before Rifai’s (1969) revision of the genus Trichoderma. Mughogho (1968) analysed the species composition of Trichoderma found in treated soils with the aid of the Rifai (1969) key and showed that several species including 7: ~~~~~~~z~~, 7: ~zar~iunum. 7: k~~?~in~~ and 7: riride were consistently present. Few studies have provided evidence to prove that 7: r!iride is a recolonizer rather than a survivor that invades and proliferates on removal of the toxicant. While acknowledging the problems of extrapolating from the laboratory to field conditions, the growth of T. r:iride sense Rifai in soils following the application of Thiram and Verdasan was most probably due to recolonization from outside the treated area. When T. hamatum SV~SURifai was similarly treated, it was found to be more tolerant of the four fungicides used. Growth of this species after treatment of soil with organo-mercurials. therefore. could be from surviving inocula and from recolonization. There is therefore a need to bear in mind the different behavioural patterns of growth of the species Trichoderrna which were previously grouped together as 7: viride by Bisby (1939) but separated by Rifai (1969). Future workers must separate the species of this genus to help gain a fuller understanding of their contribution to soil processes.
M. E. and YOUNG R. A. (1965) Changes in the soil microflora following fungicide treatment. Soil Sci. 99, 272-277. DANIELSON R. M. and DAVE%’C, B. (1969) Microbiat recolonization of a fumigated nursery soil. For. Sci. 15, 368-380. DOMSCH K. H. (1964) Soil fungicides. A. Rrr. Phytopath. 2, 293-320. DOMSCH K. H. (1970) Effects of fungicides on microbial nooulations in soil. In Pesticides in fhe Soil: Ecoloyy. b&&rion and Mowment. Int. Symp. on Pesticides in the Soil, pp. 42--46. Michigan State University. E. Lansing. &C;INS H. 0. W. and PUGH G. J. F. (1962) isolation of cellulose decomposing fungi from soil. Natlrre 1% 94-95. EVANS E. (1955) Survival and recolonization by fungi in soil treated with formalin or carbon disulphide. Trans. Br. myof. SW. 38, 335-346. FISHER R. A. and YATES F. (1963) Sr~~~i,~rjr~~Tuhks fir Ej~~~gjc~~, Agricuhrui and Metlicui Resecrrch. Oliver & Boyd. London. GRIFFITHS R. L. and MATHEWS S. (1969) The persistence in soil of the fungicidal seed dressings captan and thiram. Ann. appl. Biol. 64, 113-118. &ARSON K. C. (1973) Fungal flora and rate of decomposition or leaf litter at low temperatures. Cun. J. Soil Sci. 53, 79-84. KIMURA Y. and MILLER V. L. (1964) The degradation of organomercury fungicides in soil. J. Agric. Frf Chrm. 12, 253.-257. KREUTZER W. A. (1965) The reinfestation of treated soil. In Eco/c?y~ of Soil-hornc Plunt Parhoyrns (K. F. Baker and W. C. Synder, Eds), pp. 495-507. IJniv. California Press. KUTHUBK~THE~NA. J. and PUCH G. J. F. (1978) Effects of fungicides on the physiology of phylloplane fungi.
CORDEN
Trans. Br. my&.
ilck~~o~l~‘d~~tt~e,lt.s -.We thank the University of Malaya for a Tutorship Grant to KAJ; the University of Nottingham for the use of the grassland site; Plant Protection Ltd. for supplying the fungicides and Mr P. Smithurst for his technical assistance.
REFERENCES
AGNIHOTRI V. P. (1971) Persistence of C’aptan and its effects on microflora, respiration and nitrification of a forest nursery soil. Can. J. ~~~~~~bj~)~.17, 377-383. AC;NIHOTRI V. P. (1973) Effects of dexon on soil microflora and their ammonification and nitritication activities. Iu~. J. r.Xp. Biol. 11, 213 216. AGNIHOTRI V. P. (1974) Thiram induced changes in the soil microflora, their physiological activity and control of damping-off in chillies (Capsicrm mntm). fntf. J. mp. Biol. 12, 85-88. BISBY G. R. (1939) ~rj~~o~~~~~~~ z+ride Pers. ex Fr. and notes on H~p~c’rer~. Trans. Br. myrol. Sot. 23, 149%168. BLISS E. D. (1951) The destruction of Armilfaria mefiea in citrus soils. Phytopothology 41, 665-683. BUKCHFIEL~ H. P. (1960) Performance of fungicides on plants and in soil---physical. chemical and biological considerations. In Plarzf Pathology; an Atitmced Treurise. (J. H. Horsfall and A. E. Dimond, Eds). Vol. 3. pp. 477 520. Academic Press, New York. CARMI~HAEI J. W. (1962) Ci~r~~~porirr!n and some other aleuriosporic hypomycetes. CU~Z.J. Bar. 40, I 137- 1173. CATSKA V. and VRAXY J. (1976) Rhizosphere mycoflora of wheat after foliar application of chlorocholine chloride, urea and 4-chloro-2-mcthylhenoxyacetic acid. Folia ~~icn,hio!. 21, 268--27X
303
Sot. 71, 261-269.
LUKENS R. J. and SIXER H. D. (1958) Chemical reactions involved in the fungitoxicity of captan. Ph~topatholoq~ 48, 235-244. MOUBASH~R A. H. (1963). Selective effects of fumigation with carbon disillphide on soil fungus flora. Trrrtzs. Br. i,Z~CCli.sot. 46, 338--344. MUGHO~;H~ L. K. (1968) The fungus flora of fumigated soils. Trans. Br. J)I~CO/. Sue. 51, &l--459. PEFPLES J. L. (19741 Microbial activity in benomvl-treated soils. Phyro&doyy 64, 857 860. . Poo~t N. J. and PRICC P. C. (1971) The occurrence of C~r~.sff.~~~rjz{~ ~~~,*~~~~ in soils receiving incremental celtulose. Soit &of. Bj~~~~z~~,3, 161-166. PUGH G. J. F. and WILLIAMS J. 1. (1971) Effects of an organo-mercury fungicide on saprophytic fungi and on litter decomposition. Trans. Br. ~lyco/. Sot. 57, I64 166. RIFAI M. A. (1969) A revision of the genus Trichodermu. Mycol. Papers 116, 9-56. SAKSENA S. B. (1960) Effects of carbon disulphide fumigation on ~~jc~z~~~~~~f?~u riri& and other soil fungi. Tr~ns. Br. rrr)coi. sot. 43, 1 1 I-1 16. WAINWRIGHK M. (1977) Effects of fungicides on the microbiology and biochemistry of soils. 2. F@nzenernaehr. RodenKd. 140, 587-603. WAINWRIC~HTM. and PUGH G. J. F. (1973) The effect of three fungicides on nitrification and ammonification in soil. Soil Biol. Biochrm. 5, 577-584. WAIN~RI~HT M. and PUGH G. J. F. (1975) Effect of rungitides on the numbers of micro-organisms and frequency of cellulolytic fungi in soils. PI. Soii 43, 561 572. WARCUP J. H. (1950) The soil-plate method for isolation of fungi from soil. Nature, Land. 166, 117. WIL.LIAMS J. I. and PLJC;H G. J. F. (1975) Resistance of Chrysosporium p~nnorum to an organo-mercury fungicide. Truths. Br. m_~co/. Sot. 64. 255.-256.