J. srored Prod. Rcs. Vol. 20. No. 1. pp. 31-39. 1984 PrInted in Great Britain. All rights reserved
0022-474X/84$3.00+ 0.00 Copyright Xc 1984Pergamon Press Ltd
EFFECT OF PESTICIDES ON THE SEED-BORNE FUNGI FUNGAL SUCCESSION ON RICE IN MALAYSIA
AND
A. J. KUTHUBUTHEEN Department of Botany, University of Malaya, Kuala Lumpur 22-l 1, Malaysia (Received in final form 28 July 1983) Abstract-Three fungicides (thiophanate methyl, thiram and 2-methoxyethyl mercury chloride) and two insecticides (carbaryl and malathion) were used to study the effects of each of the pesticides on the seed-borne fungi of rice. A total of over 40 species in 23 genera of fungi. including several pathogenic forms was isolated from the untreated flowers and seeds. Fewer fungi were encountered on flowers still enclosed within the sheaths and the largest number of fungi was isolated from seeds just before harvest. Storage of the seeds for 6 months eliminated a large number of field fungi that was originally present on freshly harvested rice seeds. The fungi that persisted on the stored seeds, however, were species reported to be capable of grain deterioration and mycotoxin production. With the pesticide-treated seeds, fewer fungi were isolated from those treated with thiram or 2-methoxyethyl mercury chloride than with any of the other chemicals. Species of Curuularia were isolated as the principal fungi on seeds treated with any of the chemicals. INTRODUCTION
More than 15 years ago, Malone and Muskett (1964) rightly pointed out that there is hardly any crop of major economic importance which does not have at least one seed-borne fungal pathogen. It is evident from the published studies on the subject that rice is no exception. Neergaard (1970) listed more than 50 species of fungi of pathological significance in rice alone. Fazli and Schroeder (1966) showed the extensive presence of mycelia of Drechslera oryzae (Breda de Haan) Subram & Jain in the endosperm of rice with the hyphae of the fungus ramifying in regular patterns along the host cell wall. Even earlier, Suzuki (1930) found the fungus Pyricularia oryzae (Cav.) in the embryo, endosperm, the bran layer, kernel, glumes, and between glumes of rice, while Ganguly (1947) observed the presence of sclerotia of Alternaria padwickii (Ganguly) M. B. Ellis in the endosperm tissue of rice. While several studies have been devoted exclusively to rice diseases and their treatment (Padwick, 1950; IRRI, 1963), Ou (1972) compiled what is probably the most comprehensive treatise on rice diseases and their control. Several studies on the seed-borne fungi of rice have also placed emphasis on the major causal organisms which induce rice yellowiss hnd which produce mycotoxins (Matsumoto et al., 1955; Chung et al., 1971; Koh and Tseng, 1975; Udagawa et al., 1979). In addition to the pathogenic and spoilage fungi of rice, the saprophytic mycoflora of rice seeds also have been studied. Tzean et al. (1979) surveyed the fungal flora on rough rice and on freshly dehulled brown and polished rice in Taiwan and identified 101 species in 37 genera of fungi from the 7681 isolates they obtained. Many of these fungi were saprophytic and in several cases also probably contaminants. Kuthubutheen (1979) isolated ten species of thermophilic fungi from freshly harvested non-surface sterilised rice seeds in Malaysia. A large number of these fungi are saprophytic or only mildly pathogenic. The saprophytic fungi on rice seeds, however, do cause deterioration of the grains both in the field and during storage and hence form an important component of the seed surface mycoflora. While a vast array of recommended fungicides and other pesticides for rice exists, generally their effects have been studied only on the pathogenic fungi (IRRI, 1963; Ou, 1972; Ho, 1978; Leu and Yong, 1979). Little is known about the side effects of pesticide application on rice fungi or on the succession of fungi from before the rice flowers open until the seeds mature. The aims of this work, therefore, were to study the fungal succession of rice seeds and the effects of selected fungicides and insecticides on the incidence of seed-borne fungi of rice. MATERIALS
AND
METHODS
Pesticides and rates of application
Three fungicides, thiram (a-i. 80% w/w tetramethylthiuram disulphide), thiophanate-methyl (a.i. 70”/, w/w 1, 2-di (3-methoxycarbonyl-2-thioureido) benzene) and agallol (a.i. 4.5:/, w/w 31
A. J. KUTHUBLITHEEN
32
2-methoxyethylmercury chloride) and two insecticides: carbaryl (a.i. SSU,,w/w methylcarbamate) and malathion (a.i. 8Oob w/v 5-l , 2-di (ethoxycarbonyl) ethyl phosphorodithioate), were used in this study. The compounds were applied at manufacturers’ recommended dosages: thiram 2 g 1 ‘. thiophanate-methyl 0.88 g 1-l. carbaryl 1.55 g I-‘. and malathion 2.2 ml 1 ‘_
I-napthyl NO,O-dimethyl the following agallol 5 g 1-l.
Spruying
Rice seeds (variety SMl) were sown in plastic trays and were allowed to grow for seven days before they were transplanted into pots using three plants per pot. The pots were then arranged into plots of 20 pots each and two such replicate plots were set up for each pesticide treatment. Starting from one week after transplanting until harvest, the plants were sprayed separately with the appropriate fungicide or insecticide once every 28 days. Over the 120 days from transplanting to harvest, a total of five such sprays were applied; three (sprays I-III) before the panicles appeared, and two more (sprays IV and V) after the panicles had appeared. At each treatment, the plants in any one plot were sprayed to the point of run-off using the recommended amount of the appropriate chemical mixed in 1 1 of tap water containing 1 ml tenac sticker as a wetting agent. Control plots were also set up and the plants were sprayed with water containing 1 ml 1-l of the sticker. Sampling
Flowers and seeds from at least 10 different panicles were collected 28 days after spray III; 3. 14 and 28 days after spray IV; and 3 and 14 days after spray V. These samples are referred to as day 3, day 14 or day 28 samples respectively. On each occasion a total of not less than 300 flowers or seeds was collected for any one treatment. The flowers or seeds were washed serially in several changes of sterile distilled water and then plated on corn meal agar (CMA) and in Petri damp chambers using ten randomly selected flowers or seeds per plate and 20 replicate plates for any one treatment. The plates were incubated at 28 + 2°C and then examined after five days and subsequently at regular intervals until no new species were encountered. The number of fungi isolated from each of the 200 seeds plated out (total number of isolations) and the number of species or taxa that constituted the isolations were recorded. The moisture content at the various stages of seed development was also determined. The following four different stages were recognised during seed development: flowers before the emergence of panicles from sheaths; flowers in newly emerged panicles; milk stage: and seed set. In order to correlate the effects of pesticides with the fungi occurring at the different stages of seed development, only those flowers or seeds in the appropriate stages of development were collected. On day 28 of spray IV and on day 3 of spray V (Tables 2 and 3), for example. most of the seeds were in the milk stage although there were still panicles that had just emerged. On these occasions. however, only seeds in the milk stage were collected to investigate mycoflora associated with them. Storuge
After harvesting. one lot of the control-treatment seeds was sun-dried on brown paper for a week and the fungi occurring on these seeds were determined as described above. Another lot was sun-dried on brown paper for a week and subsequently stored at room temperature in sterile plastic bags for six months before the fungi occurring on them were determined using the method described above. The effects of storage of seeds that had received prior pesticide treatments are being investigated further. RESULTS Fungal succession,
control treutment
A total of over 40 species in 23 genera of fungi was isolated from flowers and seeds not receiving pesticide applications (Table 1). Of the fungi encountered, the pathogenic or potentially pathogenic fungi isolated included Alternaria padwickii (Ganguly) M. B. Ellis, Cercospora or_vzae Miyake, Drechslera oryzue (= Drechslera state of Cochliobolus miyabeanus (Ito & Kuribayashi) Drechsler ex Dastur). and several species of Curvulariu such as C. lunata (Wakker) Boedijn, C. lunara var.
*Aggregated.
spccxs
oxysporum
wth
pullulun~
Sacc.)
mqdicus
tlndividual
sphaerica
sp.
nor determined.
Nigrospora
Fusidium
Pifhomyces
Aureobasiditrm
(W. G. Smith) Peni
F. cubnorum
(F.
6
sp.
8
Fusurium
associated
Flowers before emergence of panicles from sheath
Mean number of species per 200 flowers or seeds
I. Fungi
Muisturc cotttent (“, fresh weight basis)
Table
species that occurred
sp.
(A.
nigert
alternaru
m&r
or flowers.
Aureobasidium
Cunningham&
pullulans
echinulata
racemosurn
moydirus
S.wcephalastrum
Pithomyces
A. jiimigarus)
(A.
A.~ppergillus sp.
sp.
maydicus
sp.
)
Rhiropus
nigricans
crrrde
globosum
orww
sp. Chueromium
Ccrcosporo
Coprinus
Trichodwvu
M. 8. Ellis
Dichoronlophrhoropsh
nw~pheorum
Miyakr
guepini
P. charrarum)
(P.
sp. halodes Pithomyces
D.
( D. orvaw
Drechslera
Pestaloliopsis
Penicillium
F. monolifbrme
Sheld.) sp* Cladosporium sp. (C. ox.wporum C. cladosporioides) Phoma sp.
F. solani
F. o.xysporum
culmorum t
(F.
C. cladosporioides)
ferreus) Fusarium
o.x,vsporum
(C.
A.
A. Jiimigatus
sp.
urriu t
t A. padwickii) Nigrospora sp. (N. sphasrica t N. orvrae (Berk. & Br.) Petch) Aspergillus sp. (A.
F. eulmorum) Cladosporium
t
rragrosridis)
Allernaria
C.
A. flaws
t
pallescms
C. .wneggolensi,c
c.
var.
sp.
lunarat
25
Seed set harvest)
Ibefore
of occurrence
C. lunutu
(C.
Currularia
frequency
F. sol&
oxysporum
sp.
(F.
nigricans
Rhi:opus
Stey.
Fusarium
P. wrsicolor)
sp.
sp.’
cucurbirarum
sphaericu t
guepini
(Desm.)
(P.
Ptwaloliop.sis
Penicrlhum
Choanephoro
Nigrospora
C. senegalensis)
19
49
Milk stage
in order of decreasing
sp. (C. lunara t c. pollesrens
Curwlaria
of rice reeds arranged
on at lsazt 60^, of the ,eed,
niger lunara
Curwlaria
cucurbitorum
spharrica
Aspergillus
Choonephora
Nigrosporo
pullulun.~
8
12
Flowers m newly emerged panicles
stages m the development
Aurrobasidium
the various
(Rand)
34
A.
Table
2. Mea”
“umber
J.
KLITHUBLJTHEEN
of fungifor
ofapec~s
201) flowers Spray
or seeda l’ollowmg
5
Day
Day
IJ
2X
3
I4
X
IX
II)
32
IX
27
I6
Thlophanate-Me
5
I +
6
1+375)
5 16.7) ‘I
( -33.3) >*
1
33.3)
(
Ag”llOl
Flowers
Stage
6
4
(
I
25.91
( -6.2) Y*
I5
II
, -- I6 7) Thlrdm
l?.j)
Day
( 10.0) (~ 15.6) 20 30
-11.1)
I’) 1+5hl
> lh.7)
v
Da!
( , -3.33) I
apphcation
Spray
x Malathion
pesnude
IV
I’)*
( -~52.0) (40.6) 8* 5* , - 75.0) ( - 75.0)
I 167) 9* , i(l 0) X’
4*
3*
( ~ 55.51 f-850)
50.(l)
MilL
below
(-X7.5) Seed
stage
set
emergence of panicles
(befwe
frclm
harvest)
lower
than control
in parenthesis
respective
control
shou
(P = 0.05).
the percentage
~ncreaac t + 1 or
decrease
( - ) compard
to
the
value.
aeria (Batista, Lima & Vasconcelos) M. B. Ellis, C’. pallrscens Boedijn, C. senegalensis (Speg.) Subram., and C. erugrostidis (P. Henn.) J. A. Meyer. The blast fungus, Pwicularia oryzae Cav., however, was not encountered. From the time the flowers emerged till harvest, there was a gradual increase in the number of species and a 15fold increase in the number of isolations encountered on the seeds (Tables 2 and 3). Species of Fusarium and Penicillium which were the most frequently encountered fungi on flowers within sheaths and on those that had emerged were later replaced, however, by species of Nigrospora and Curwluriu as the predominant fungi on seeds in the milk stage. Aureobasidium pullulans (De Bary) Arnaud which was common on flowers was not encountered on the fully-formed seeds. The milk stage had the highest moisture content and was characterised by the presence of zygomycetous forms such as Choanephoru cucurbiturum (Berk. & Rav.) Thaxt., Rhizopus nigricans Ehrenb., and, to a lesser extent Syncephulustrum rucemosum Cohn ex Schroet. and Cunninghamella echinata (Thaxt.) Of these fungi. only R. nigricans persisted on the later stages of the seeds. With seed set, species of Aspergillus (A. nigrr van Tiegh.; A.,fitmigutus Fres.) and Alternuria (A. alternata (Fr.) Keissler; A. padwickii) which were absent or infrequent up till the milk stage, were encountered more frequently on the fully-formed seeds. Species of Aspergillus and Alternaria and those of Curzwluriu, Nigrosporu and Fusurium together constituted the principal fungi on rice seeds just before harvest.
Spray
v
Da)
Day
2X
3
Da) I4
(‘ontrc,l
IXX4
I Y5?
2836
Carbaryl
I YXO
I616
I
(+51)
3856t
( + 36.0)
1721
IhlI8 I646 c I-u%,, c 15.7)
Malathion
I 6%
Thiophanatr-Me
I ThlWm
1792
924’
1+31.2
LOX*
-51
,
-61.7)
I
(II
Milk
1444*
74.0)
l-49.1) 624*
274*
hot(* stage
37110t
I 8.2)
111.6)
, Agallol
3401(t ( +x.7)
I X6.0) \tagc
I
7x 0)
Seed set (hefore harvest
*SlgniRcantly Figures
lower
in parenthesis
COntrol
value%
than
control
(P = 0 115). Wgnilicantly
hhow percentage ~“crcasz
higher
than
control
I + 1or decreaw t ~~) compared
I
(P = 0.05).
to the respective
Effect of pesticides on the seed-borne fungi
35
EfSect of the insecticides
The application of either of the insecticides did not cause a significant reduction in the number of species encountered on the flowers or seeds compared with the control (Table 2). Both in terms of numbers and composition, the species of fungi isolated from the treated seeds throughout the study remained somewhat similar to those of control. The total number of isolations on the insecticide-treated flowers attached to panicles still enclosed within the sheaths, however, were significantly lower than those of control (Table 3). Once the flowers had emerged from the panicles and up to the milk stage, the application of the insecticides did not significantly alter the number of isolations on the seeds. On day 14 of spray V, however, there was a significant increase in the number of isolations encountered on the treated seeds just before harvest. In terms of species composition of insecticide-treated seeds, there was no noticeable difference compared to untreated seeds. Particularly after seed set, the principal fungi occurring on the insecticide-treated seeds were species of Curvularia (C. lunata, C. lunata var. aeria, C. senegalensis, C. pallescens, C. geniculata, (Tracy & Earle) Boedijn, C. eragrostidis, C. intermedia Boedijn and C. clavata Jain in that order), Alternaria (A. padwickii, A. alternata, A. tenuissima (Kunze ex Pers.) Wilt.), Nigrospora, Aspergillus and Fusarium. Curvularia formed the bulk of the isolations on the insecticide-treated seeds and C. lunata, C. lunata var. aeria, C. senegalensis, C. pallescens and C. eragrostidis in particular were encountered on more than 60% of the insecticide-treated seeds. Also C. geniculata and C. clavata were absent on control seeds but appeared on the insecticide-treated ones. Eflect af the fungicides
(a) Thiophanate-methyl. After spray IV of thiophanate-methyl, there was no significant difference in the number of species encountered on the treated seeds. However, after spray V of the compound, there was a significant decrease in the number of species encountered on the treated seeds (Table 2). It appears, therefore, that those species that occurred during the early stages of seed development were not affected by the fungicide while more than 40% of the species that were associated with the later stages of seed development were susceptible to the compound. It is also evident from the significant increase in the number of isolations of day 14 of spray V (Table 3) that those species of fungi that persisted on the treated seeds occurred on a larger number of seeds following the application of thiophanate-methyl. The fungi that persisted on the thiophanatemethyl-treated seeds were mainly Curt&aria (C. lunata, C. lunata var. aeria, C. pallescens, C. eragrostidis, C. senegalensis, C. geniculata), Alternaria (A. padwickii, A. alternata), Rhizopus nigricans, Nigrospora sphaerica (Sacc.) Mason, Pithomyces (P. chartarum (Berk. & Curt.) M. B. Ellis, P. maydicus (Sacc.) M. B. Ellis). and Drechslera (D. oryzae, D. halodes (Drechsler) Subram.). (b) Thiram. Compared to the control, consistently fewer species of fungi were isolated from
thiram-treated flowers and seeds (Table 2). The deleterious effects of thiram were particularly noticeable from day 28 of spray IV to day 14 of spray V. After spray V, only 257; of the number of species occurring on control seeds in the later part of the milk stage and on control seeds just before harvest persisted on the treated seeds. In day 3 samples of spray IV and V, the number of isolations obtained from thiram-treated seeds were only about 30% of the respective control values (Table 3). On all other samples, the reduction in the number of isolations was between 40-50% compared to the control. On thiram-treated seeds, there was an abundance of Curvularia (C. lunata, C. eragrostidis, C. pallescens, C. senegalensis, C. geniculata, C. lunata var. aeria), Alternaria (A. alternata, A. padwickii), Nigrospora sphaerica, Fusarium (F. oxysporum Sheld. emend Snyd. & Hans.; F. solani (Mart.) Appl. & Wr. emend Snyd.), and Penicillium sp. Species OFPenicillium were isolated in much higher frequencies of occurrence on thiram-treated seeds than on the others. (c) Agallol. As with thiram-treated flowers and seeds, consistently fewer species of fungi compared to the control were encountered on agallol-treated flowers and seeds of rice (Table 2). The effects of agallol, however, were more deleterious compared to that of thiram and after spray V, only about 15% of the number of species occurring on control seeds were isolated from the agallol-treated ones. Similarly, after spray V of agallol, only about 15-20% of the total number of isolations obtained from the control seeds were encountered on the treated ones (Table 3). The
A. J. KUTHUBUTHEEN
36
3-4 species of fungi that persisted on the seeds after spray V of agallol were drawn mainly from among the following: Curvularia (C. lunata, C. eragrostidis, C. pallescens), Fusarium oxysporum, Alternaria alternata and Aspergillus niger. The deleterious effects of the three fungicides towards fungi colonizing flowers and seeds of rice were in the order agallol > thiram > thiophanate-methyl.
Curvularia
sp.
Species of Curvularia taken together were the principal fungi on seeds treated with any of the five pesticides used in the study. Evidently the Curvularia sp. were not only capable of withstanding any of the pesticides but they also rapidly recolonized the treated seeds. This observation is significant especially in view of the fact that several species of Culvularia are known to be pathogenic or at least potentially so.
Storage oj‘ji-eshly harvested seeds After harvesting and drying, the moisture content of the seeds was 147; (fresh weight basis) compared to the 25% at the time of seed set before harvest. Accordingly, the mean number of species per 200 seeds after harvesting was only 21 compared to 32 before harvest (Table 4). Species of Curvularia and Alternaria that were more frequently encountered on seeds before harvest were not as abundant as species of Aspergillus on harvested sun-dried seeds. Storage of the seeds resulted in a further decline in the number of species of fungi encountered on the seeds. The principal fungi on stored seeds were essentially species of Aspergillus (including A. j?avus Link), Penicillium (including P. islandicum Sopp.), and to a lesser extent Chaetomium (C. globosum Kunze ex Fr., C. cochlioides Palliser) and Rhizopus nigricans. Nevertheless, it was evident that the mycoflora of stored rice seeds comprised of fungi reported to be cellulose and starch deteriorating fungi as well as those potentially capable of mycotoxin production. Table 4. Fungi associated wth freshly harvested seeds and with seeds stored for 6 months Freshly harvested (sun-dried only)
Seeds after harvest (sun-dried arld atored for 6 months)
14
12
Mmsture content (I’,, fresh weight basis) Mean number of species per 200 seeds
IO
?I Aspergillus (A.
sp.
A.spargi/lus (A
niger
A. fk~rrau~
niger
A. ,fumigutus
.4. .jmus) sp.*
A. fumigatus)
sp.
Curwlaria
sp.
Penicillium
lunatu c‘. erugrostirli.5
(C.
Chorron~ium
C‘. rochlioides
C. intwmdru
C‘.oerruc(formi.s (A.
Agarwal & Sahni)
sp.
Altrrnario
(c‘.
A. tenuissrmu Penicillrum
sp*
Drechsleru
oryxo
sp.
F. oxy.~porum
(N.
atro
) Corda
sp.
sphaericu
N. orvxze) Pewalotiopsrs
gwpini
Phoeotrrchoconis
croralorrur
(Salam & Rae) Subram. Rhixpus
nigricans
*Aggregated. species not determined. CMA and Petri damp chambers: 28 + 2 C.
lunatu
n. oi-r:ue
I
(F. culmorum
Nigrospora
)
nigric uns
C‘. rragmstidis
A. padwirkir
Stachybotrw
Rhimpus
Curaularia sp.
altrrnatu
Fusnrium
sp
( C. globmum
(‘. pallr.wn.\
)
Effect of pesticides
on the seed-borne
fungi
37
DISCUSSION
Christensen (1965) categorised fungi that invade grains of cereals into three ecological groups, namely field fungi, storage fungi, and advanced decay fungi. The field fungi, including species of Alternaria, Fusarium, Drechslera and Cladosporium. require a relatively high moisture content of 22-252; on a wet weight basis and invade developing grains, kernels, or even matured seeds before these are harvested. These fungi do not occur after harvest essentially due to a decline in the moisture content of the seeds. The storage fungi including species of mainly Aspergillus and Penicillium, invade cereal grains after harvest and during storage and they were shown to tolerate relatively low moisture contents and dry conditions (Christensen, 1965). In the present study, a similar distinction was observed between the field and storage fungi. The field fungi of rice, however, showed discernible patterns of succession with Fusarium, Penicillium and to a lesser extent Aureobasidium pullulans occurring on the flowers and Curvularia, Nigrospora, Alternaria, Fusarium and to a lesser extent Cladosporium occurring more frequently on the seeds before harvest. The question of the origins of the fungi on flowers enclosed within sheaths, however, becomes an interesting one. While Aureobasidium pullulans has been shown to be endophytic in other plants (Pugh and Buckley, 1971). there appears to be no findings of it being so in rice. The other species that occurred on flowers within the sheaths include dry-spored fungi such as Penicillium, Pithomyces and Nigrospora which could have been drawn into the sheaths on films of water. Fusarium which is predominantly splash dispersed could also have been brought into the sheaths by run-offs from leaves and stalks. Although it is probable that many other fungi could be similarly brought into the sheaths, the low moisture content and the relative non-availability of nutrients in the young flowers could make it unfavourable for the growth of these other fungi. Storage of seeds caused a reduction in the number of fungi on rice seeds. The fungi that persisted included species of Aspergillus, Penicillium, Chaetomium, Curvularia and Drechslera. Neergard (1970) showed that, on the whole, storage of seeds caused a reduction in the amount of seed-borne inoculum. He showed that while many species have reduced vitality within a year of storage, D. oryzae showed no obvious reduction in vitality even after 2 years. Tzean et al. (1979) showed that the field fungi on rough rice did not change their prevalence significantly over 2 years of storage. They observed, however, that there was reduction in the number of field fungi and a corresponding increase of storage fungi on brown and polished rice stored for longer periods in polyethylene bags. The most commonly encountered storage fungus in their study was Aspergillus. Christensen and Lopez (1965) reported that the storage of rough rice at a moisture content of 13.0-14.8% at 22-28” for 98 or 413 days resulted in the death of field fungi. Their findings and those of Fanse and Christensen (1966) and Christensen (1969) differ from those of Tzean et al. (1979) for rice or Wallace et al. (1976) for wheat. In the present study, there was a marked reduction and not a total elimination of fungi on seeds stored over a period of 6 months. Tzean et al. (1979) attributed such differing results and observations to variations in methods of isolation and in the recording of incidence of fungi on seeds. It is evident, however, that variations are bound to occur essentially because of the different rice varieties used and because of the different strains and physiological races of the same species of fungi in geographically separated localities where the studies have been carried out. In the present study, three fungicides (thiophanate-methyl, thiram, agallol) and two insecticides (carbaryl, malathion) were used to investigate the effects of each of the compounds on the seed-borne fungi of rice. The deleterious effects, if any, of insecticides towards fungi associated with aerial parts of plants have been reported to be only minimal (Hislop, 1976). In the present study, it was evident that the insecticides used, carbaryl and malathion, did not eliminate any of the fungi found on flowers or seeds; instead, only the frequency of occurrence of these fungi was sometimes altered. The application of fungicides to soil and on leaf surfaces have been shown to reduce the number of fungi associated with these substrates (Klincare et al., 1971; Hislop, 1976; Kuthubutheen and Pugh, 1979). In the present study, a similar effect was observed on the rice seed surface when the fungicides thiophanate-methyl, thiram and agallol were used. The increase in fungal numbers in soil following fungicide application has been attributed by Ko et al. (1971) to the increased availability of ammonium-N in the treated soil. Whether this is the case on the seed surface, is at this point only a matter of speculation.
38
A. J. KUTHUBUTHEEN
Of the three fungicides used, the systemic compound thiophanate-methyl produced the least deleterious effects on the numbers of seed-borne fungi of rice. The fungitoxicity of thiophanatemethyl may be attributed to the heterocyclic transformation of the compound to the benzimidazole ring (Woodcock, 1972) and hence the compound behaves like any other benzimidazole compound such as benomyl or thiabendazole. Dickinson (1973) and Warren (1974) have shown that systemic fungicides have less deleterious effects on the saprophytic fungi, and Edgington et al. (1971) showed that benomyl and thiabendazole were not deleterious towards fungi such as Alternaria, Curvularia and Drechsfera. In the present study thiophanate-methyl similarly did not eliminate these fungi occurring on rice seeds. Although agallol itself is not normally recommended for rice, several organomercury compounds are widely used as seed protectants of rice and other cereals. In a preliminary study, Kuthubutheen (1980) showed that agallol-treated rice plants were stunted and the weight of seeds produced was negligible. Klincare et al. (1971) showed that the mercurial seed-dressing, mercuran, reduced the number of epiphytic micro-organisms on seeds of barley and lucerne by about 72 and 45% respectively compared to the control. The decrease in the epiphytic microflora of seeds caused by mercuran and other seed dressings was also shown to produce changes in the composition of microflora above and below ground during the initial stages of plant growth and development. In the present study, agallol produced the most deleterious effects on the seed-borne fungi of rice. Agallol is a methoxyethylmercury compound and as early as 1944, Booer had shown that such radicals form highly ionized salts that are more soluble and volatile and hence more fungitoxic compared to the phenyl and tolyl radicals. The persistence of thiram (tetramethylthiuram disulphide) which is used as a seed treatment against damping off diseases has been examined by Richardson (1954) who found that whilst thiram rapidly disappeared from compost soil, it persisted for over two months in sandy soil. The fact that seedling protection was subsequently observed after the disappearance of the fungicide pointed to the possibility of active degradation products. Hylin and Chin (1967) used 35S- and 14C-labelled compounds and noted the formation of carbon disulphide, dimethylamine and four unidentified metabolites in addition to fi-glucoside and alanine conjugates. Carbon disulphide and dimethylamine, which are themselves fungitoxic, may account for the persistence of the fungitoxic effects. Klincare et al. (1971) showed that 507; tetramethylthiuram disulphide reduced the number of epiphytic micro-organisms on seeds of barley and lucerne by about 79 and 36% respectively compared to control. In the present study, thiram reduced the number of species of fungi on rice seeds just prior to harvest by 75% compared to control. The persistence of the fungitoxic effects of thiram from day 3 to day 14 of spray V, for example, indicate that active degradation products of thiram may probably be influencing the establishment of fungi on seeds still attached to the plants. Although fungicides eliminate a large number of seed-borne fungi, those species that persist include organisms known to be capable of grain deterioration and toxin production. An increased incidence of species of Curvuluria stands out as one of the more significant side effects of pesticide applications. A systematic study of the effects of storing pesticide-treated seeds would contribute significantly to our knowledge of the roles and survivability of seed-borne fungi. Acknowledgements-The author is grateful for the rice seeds, and Mr S. Vythilingam
to Professor T. A. Oxley, University for technical assistance.
of Aston for advice on the paper.
MARDI
REFERENCES Booer J. R. (1944) The behaviour of mercury compounds in soil. A. uppl. Biol. 31, 346359. Christensen C. M. (1965) Fungi in cereal grains and their products. In Mycotoxins in Foodstuffs (Edited by Wogan G. N.), pp. 9-14. M.I.T. Press, Cambridge, Mass. Christensen C. M. (1969) Influence of moisture content, temperature. and time of storage upon the invasion of rough rice by storage fungi. Phytopnthology 59, 145-148. Christensen C. M. and Lopez L. C. (1965)Relation of moisture content and length of storage to changes in the microflora and germination percentage of rough rice. Phytopathology 55, 935-956. Chung C. H.. Ling K. H., Tung S. S. and Tung T. C. (1971) Study on fungi of the stored unhulled rice of Taiwan. J. Formosan med. Ass. 70, 258-266.
Effect of pesticides
on the seed-borne
fungi
39
Dickinson C. H. (1973) Effects of ethirimol and zineb on phylloplane microflora of barley. Trams. Br. mycol. Sot. 60, 42343 1. Edgington L. V.. Khew K. L. and Barron G. L. (1971) Fungitoxic spectrum of benzimidazole compounds. Phytopathology 61, 42-44. Fanse H. A. and Christensen C. M. (1966) Invasion of rice stored at moisture contents of 13.5 to 15.59,. Phyropatholog~ 56, 1162-l 164. Fazli S. F. I. and Schroeder H. W. (1966) Kernel infection of Bluebonnet 50 rice by Helminthosporium oryzae. Phvtopathology 56, 507-509. Ganguly D. (1947) Studies on the stackburn disease of rice and identity of the causal organism. J. tndiun bot. Sot. 26, 233-239. Hylin J. W. and Chin B. H. (1967) Delayed phytotoxicities produced by dithiocarbamate fungicide residues. I&. Gong. Pl. Prot.. 6th I’iennu, p, 614 (abstr.). Wien. med. Akad.. Wien. Hislop E. C. (I 976) Some effects of fungicides and other agrochemicals on the microbiology of aerial surfaces of plants. In Microbiology of Aerial P/ant Surfuc~s (Edited by Dickinson C. H. and Preece T. F.), pp. 41-74. Academic Press, London. Ho B. L. (1978) Fungal diseases of rice in Malaysia. In Proc. Rice Rerieu, Meeting (Edited by Ismail A. A., Varughese J. and Abdullah W. R.), pp. 240-248. MARDI Res. Station. Bumbong Lima. IRRI (International Rice Research Institute) (1963) The Rice Blasf Fungus. 507~~. John Hopkins Press, Baltimore. Klincare A. A.. Kreslina D. J. and Mishke I. V. (1971) Composition and activity of the epiphytic microflora of some agricultural plants. In Ecology of Leqf Surface Microorganisms (Edited by Preece T. F. and Dickinson C. H.), pp. 191-202. Academic Press, London. Ko W. H., Hora F. K. and Herhcska E. (1971) Isolation and identification of a volatile fungistatic substance from alkaline soil. Phytopatholog_v 64. 1398-1400. Koh H. L. and Tseng T. C. (1975) Isolation and identification of an aflatoxin-producing strain of Aspergillus,flaLlus from stored rice. Bot. Bull. Acad. sin. 16, 115- 125. Kuthubutheen A. J. (1979) Thermphilous fungi associated with freshly harvested rice seeds. Truns. Br. m~col. Sot. 73, 357-359. Kuthubutheen A. J. (I 980) Effect of pesticides on leaf and seed surface fungi of rice. In Tropicul Ecology and Decrelopmenr: Proc. Ini. Svmp. Trap. Ecol., 5th. Kuala Lumpar, 1979(Edited by Furtado J. I.), pp. 1021-1028. Int. Sot. trop. Ecol.. Kuala Lumpar. Kuthubutheen A. J. and Pugh G. J. F. (1979) Effect of fungicides on soil fungal populations. Soil. Biol. Biochem. 11, 297-304. Leu L. S. and Yong H. C. (1979) Comparative effectiveness of IO recommended fungicides in rice blast disease (Pyricularia orvxze) control. PI. Prot. Bull. 21, 415422. Malone J. P. and Muskett A. E. (1964) Seed borne fungi. Proc. int. Seed Test. Ass. 29, 179-384. Matsumoto K., Cheng C. C., Wu L. C., Wang M. C., Su H. J.. Lee C. H. and Chen P. C. (1955) Monthlv Report of the PhJjtopathological Luboratory on Deteriorated Rice. National Taiwan University, 234 pp. Neergaard P. (1970) Seed pathology of rice. In Plant Disease Problems: Proc. Int. Symp. PI. Pathol., Ist, Delhi, 196667 (Edited by Raychaudhuri S. P. et al.), pp. 57-68. Indian phytopath. Sot.. New Delhi. Ou S. H. (1972) Rice Diseases and Pests, 368 pp. Commonw. mycol. Inst., Kew, Surrey. Padwick G. W. (1950) Manual of‘ Rice Discuses. 198 pp. Commonw. mycol. Inst.. Kew, Surrey. Pugh G. J. F. and Buckley N. G. ( 1971) Aureobasidium ptdlulons: an endophyte in sycamore and other trees. Trams. Br. mycol. Sot. 57, 227 -23 1. Richardson C. T. (1954) The persistence of thiram in soil and its relationship to the microbiological balance and damping-off control. Can. J. Bot. 32, 335-346. Suzuki H. (1930) Experimental studies on the possibility of primary infection of Pyricularia oryzae and Ophiobolus miyabeanus internal of rice seeds. A. phvtopath. Sot. Japan 2, 245-275 (original not seen, cited by Neergaard 1970). Tzean S. S.. Shu M. C. and Kuo T. T. (1979) Influence of storage regimes upon the population dynamics of fungi on the stored rice. Bot. Bull. Acad. sin. 20, 58-88. Udagawa S., Muroi T.. Kurata H., Sekita S.. Yoshihira K. and Natori S. (1979) Chuetomium udagawa, new species: A producer of sterigmatocystin. Trans. mycol. Sot. Japan 20, 475-480. Wallace H. A. H., Sinha R. N. and Mills J. T. (1976) Fungi associated with small wheat bulks during prolonged storage in Manitoba. Can. J. Bot. 54. 1332-1343. Warren R. C. (1974) Differential effects of fungicides on phylloplane fungi isolated from oak. Truns. Br. mycol. Sot. 62, 215-218. Woodcock D. (1972) Structure-activity relationship. In S,vstemic Fungicides (Edited by Marsh R. W.). pp. 34-85. Longmans, London.