- Email: [email protected]
Fusarium species in soil, thatch and crowns of Poa pratensis turfgrass treated with fungicides
sorl Brv, Bj~~~,ll~,~.VOI, Ii. pp. 355 to 363 C Pergamon Press Ltd 1979. Printed rn Great Britatn
SPECIES IN SOIL, THATCH AND CROWNS OF POA PRATENSIS TURFGRASS TREATED WITH FUNGICIDES
FUSARIUM
R. W.
SMILEY
and M. M.
CRAVEN
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Accepted 10 March 1979) Summary-Little information exists concerning the effects of long-term fungicide programs on the qualitative and quantitative characteristics of fusaria in turfgrasses and in turfgrass soils. These effects were studied on Pea pratensis L. turfgrasses at three locations, using 14 fungicides, one nematicide, and five mixed fungicide programs. Some fungicides increased the numbers of fusaria in soil and thatch, some had no effect, and others greatly reduced the numbers. Changes in Fusarium species’ compositions occurred independently from the changes in propagule numbers. The proportion of Fusarrum-colonized turfgrass crowns was generally higher in fungicide-treated than in nontreated turfgrasses.
lNTRODUCTION
Fus~rium species are among the most abundant fungi in grassland soils, and they are invariably associated with the roots of Gramineae. The dominant fusaria in grasslands include F. oxysporum, F. solani, F. tri-
cinctum, and various species within the compositegroup F. roseum emend. Snyder & Hansen (Clark and Paul, 1970; Grpurt and Curtis, 1957; Warcup, 1951). In a survey of the fusaria in crowns and roots of apparently healthy Poa prutensis L. (Kentucky bluegrass) being produced for sod, Cole et al. (1973) isolated F. roseum and F. tr~ci~c~m from 100% and 37”/, respectively, of ah fields sampled. Kommedahl and Siggeirsson (1973) have also isolated fusaria from 69 of 71 fields surveyed in Iceland, with the F. roseumtypes being the species most frequently isolated from the roots and from the soil. The second most abundant species on roots, and third-ranked in soil, was F. oxysporum. Roots often also yielded F. tricinctum. Fusarium species are undoubtedly a very important component of grasstand ecosystems (Domsch and Gams, 19721, and their dominance over other fungi is greatest on senescing roots (Waid, 1957). Kentucky bluegrass is one of the most widely used turfgrasses in the United States. One of the important factors limiting its successful culture is a crowii rot named Fusarium blight (Couch and Bedford, 1966; Endo, 1961). Although the etiology of Fusarium blight is not understood, the incitants have been considered to be F. roseum f. sp. cereaiis (Cke.} Snyd. & Hans. “Culmorum” and F. rricinctum f. sp. pose (Pk.) Snyd. & Hans. (Couch, 1974). These and other fusaria are generally associated with senescing leaf tissues on diseased plants, and are also often isolated from decaying plant crowns. Fusarium blight is prevalent only on older stands of Kentucky bluegrass that are highly maintained as golf course fairways, home lawns, or commercial building landscapes. A characteristic of the intense management given to these areas is the routine application of pesticides. The influence of fungicides on the Fusarium species’ distribution in turfgrasses has
not been investigated. An understanding of this interaction is especially important on Fusarium blightprone turfgrass sites, for if a fungicide that is regularly applied to control another disease leads indirectly to an increase in the inoculum potential of pathogenic fusaria, that fungicide’s use could be counter-productive to subsequent Fusarium blight control measures. Knowledge about fungicide-Fusarium interactions is also basic to achieving an understanding of the etiology of Fusarium blight. We report the occurrence of Fusarium species on golf course fairways where Fusarium blight control investigations have been conducted (Smiley and Craven, 1977; Smiley et al., 1978), and on long-term plots where fungicides’ effects on nontarget soil organisms and on microbial processes are being studied (Smiley and Craven, 1978, 1979a). MATERIALS AND METHODS
Disease control studies on Long ls~~nd
Fungicides were evaluated during 1976 and 1977 on 12-yr-old Kentucky bluegrass fairways at the Mill River Club, Oyster Bay, NY. Plots were established on fairway areas that had been uniformly affected by Fusarium blight the previous year, and they were given the golf course’s complete fairway maintenance program, including applications of fungicides for control of other diseases. None of these fungicides appeared to alter the incidence of Fusarium blight. In 1977, another plot was established at the Nissequogue Golf Club, St. James, NY, where Fusarium blight had occurred on a IO-yr-old Kentucky bluegrass fairway in 1976. The Mill River fairways are routinely watered with automatic irrigation systems, whereas those at Nissequogue are not. Experimental designs were randomized complete blocks with four replications per treatment. Applications of fungicides began about 20 May each year, and all golf-course resuhs reported here are from treatments in which fungicides were drenched into the thatch or soil with supplemental sprinkler irrigation
R. W. SMILEY and
356 Table
1. Pesticide
names*
M. M. CRAVFN
and application
rates on Long
Island
golf courses
Applications
(kg a.i. ha-
1976 Treatment code BF
I Mt Td GA * Chemical
Trade Common
name
benomyl iprodione methyl thiophanate triadimefon (none) names
are the same
1977
(?
June 7
June 21
Aug. 2
May 24
June 12
July 11
Tersan 1991 (50) Chipco 26019 (50) Spot Kleen (70) Bayleton (SO) CGA 64251 (21.5)
11.2 11.2 0 0 0
11.2 11.2 0 0 0
6.6 6.6 0 0 0
0 11.2 11.2 3.0 2.5
0 11.2 11.2 3.0 2.5
0 11.2 11.2 3.0 2.5
as those
name
’)
listed
in Table
2, except for triadimefon, which is Names for CGA 64251 are
1-(4-chlorophenoxy)-3,3-dimethyl-1-( lH-1,2,4-triazol-1-yI)-2-butanone. confidential.
and immediately after applications. Details of applications, plot areas, and additional treatments are given by Smiley and Craven (1977) and by Smiley ef al. (1978). Treatments reported here are summarized in Table 1. During the last week in August each year, seven cores, of 2.5 cm dia x 2 cm, with thatch intact, were collected from random locations within each treatment area. Cores were placed in plastic bags and were refrigerated during transport to the laboratory. The cores were then chopped, mixed thoroughly, and log of each thatch + soil mixture were suspended in 95 ml 0.1% water agar in a 200 ml milk dilution bottle containing 250 (3 mm dia) glass beads. A portion of the remaining thatch + soil was dried to permit adjustment of the data to an oven-dry soil basis. The suspensions were then placed horizontally in a reciprocating shaker and shaken vigorously for 10 min at 200 cycles min-‘. The suspension was diluted in a lo-fold dilution series in O.l:/, water agar, and 1 ml of an appropriate dilution was dispensed aseptically onto plates of peptone-PCNB-agar medium (Nash and Snyder, 1962). After incubation of the plates for 4 ~7 days at 25”C, the fusaria were counted and propagule numbers were adjusted to an oven-dry soil basis. Fusarium isolates were then transferred from the Nash and Snyder medium to plates of potato-dextrose-agar (PDA) and of carnation-leaf-agar (CLA) (Toussoun and Nelson, 1976). Plates were incubated together at about 25°C under fluorescent lights for 410 days. Fusarium species were identified by observing macroscopic characteristics of PDA colonies and microscopic details from colonies growing on CLA; the taxonomic system of Booth (1971) was used throughout this study. Many representative isolates were submitted to the Fusarium Research Center, The Pennsylvania State University, for confirmed diagnoses by Drs T. A. Toussoun and P. E. Nelson. The taxonomic system of Booth does not recognize F. robustum as a species distinct from F. yraminearum. The separation was made in our study in view of its prevalence in the turfgrasses of New York State. Gerlach (1977) described similar isolates as F. robusrum and later, utilizing isolates from the Mill River Club, which were submitted for comparison by Dr T. A. Toussoun, showed that these were also similar to F. robustum. Personal communications with Dr P. E. Nelson indicate that isolates similar to F. robusduring
turn were also shown by Dr Lester Burgess to be common in some pasture soils in Australia, and that they were considered by Dr C. M. Messiaen to be a “soiltype of F. graminearum” (Messiaen and Cassini, 1968). The species designation F. robustum was, therefore, selected in our study as a supplement to the taxonomic system of Booth (1971). Identifications in 1976 were made for 100 randomly-selected isolates per fungicide treatment, and in 1977, to exclude the possibility for non-randomlybiased selections of Fusarium isolates, every isolate from the Nash and Snyder medium was identified; the numbers of identifications per treatment ranged from 39 to 110 (average of 59). Fusarium species were also isolated from the crowns of all individual tillers present in the 1976 and 1977 core samples. Foliage and roots were trimmed from coronal tissue, and the crowns were then placed in cheesecloth bags and washed under rapidly-running tapwater for 223 h. Crowns were blotted dry on clean paper towels, soaked in a 1 :l mixture of sodium hypochlorite (5.25%) and ethanol (95%) for 1 min. rinsed in distilled water, and then spaced individually on acidified PDA medium (pH 5.0). Numbers of inspected crowns ranged from 25 to 35/treatment in 1976 and from 65 to 95 in 1977. Crowns colonized by Fusarium roseum emend Synd. & Hans. were enumerated but identified by cultivar name. Nontarget
organism
study ut lthuca
A 2-yr-old Kentucky bluegrass turf was installed as sod in 1975 at Cornell University. Repeated applications of fungicides were then made to replicated plots (1 x 5 m) during 1975, 1976, and 1977. Details of the plots’ management, the soil, and the fungicide application equipment have been described by Smiley and Craven (1978). The nonirrigated turf was maintained at a height of 4.4 cm, and clippings were removed during mowing. Fourteen fungicides and one nematicide (Table 2) were selected as representatives of those likely to be used commercially. Foliar spray treatments were made nine times annually, every 21 days, from April until September. Exceptions to this schedule included: (1) benomyl, applied as nine foliar applications for one treatment, and as two drenching applications in June for another treatment: (2) fenamiphos applied
Actidione RZ (0.75 + 75)
Bromosan (17 + 50)
MF 598 (15 + 60)
quintozene (or PCNB) thiram cycloheximide + quintozene
ethyl thiophanate + thiram
methiophanate + maneb
Q
EtT
MtM
(as above; alternated) (as above; alternated) (as above; CH & MtM alternated, plus F) (as above; alternated) above; EtT, CH, & MC alternated, plus Q & E)
Terraclor (75) Tersan 75 (75)
* Drenching applications. All other applications were foliar.
CH/A CH/MtM CH/MtM + F EtT/CH/Mc EtT/CH/Mc + Q + E (as
CYQ
T
Dithane M-45 (80)
mancozeb
MC
Dyrene (50) Tersan 1991 (SO) Tersan 1991 (50) Cadminate (60) Captan (SO) Daconil 2787 (75) Actidione TGF (2.1) Koban (65) Nemacur (15) Chipco 26019 (50)
Trade name (% a.i.)
anilazine benomyl benomyl cadmium succinate captan chlorotbatonit cycloheximide ethazole fenamiphos iprodione
Common name
A Bf Bd CS CP CH CY E F I
Treatment code
514 514 514 + 1 3/3/3 31313 +9+9
9
9
9
(as (as (as (as (as
above) above) above) above) above)
2.3 + 12.2
3.1 + 9.2
0.1 + 1.7
6.9 9.2
9.8
9 9 9
2: 3:1
9 9 2* 9 9 9 9 9 1* 9
7.7 3.1 12.2 3.7 7.7 9.2 0.1
Annual applications Number kg ha-’ appl-’
dimethyl 4,4-a-phenylenebis[3-thioallophanatelplus manganese ethylenebisdithiocarbamate -
diethyl 4,4-u-phenylenebis[3-thioallophanatelplus bis(dimethylthiocarbamoyl)disulfide
~-[2-(3,5-dimethyl-2-oxocyclohexyl~2-hydroxyethyl] glutarimide plus pentachloronitrobenzene
2,4-dichloro-6-(o-chloroanilino)-s-triazine methyl I-(butylcarbamoylf-2-benzimidazolecarbamate methyl I-(butylcarbamoyl)-2-benzimidazolecarbamate cadmium succinate N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide tetrachloroisophthaionitriie ~-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide 5-ethoxy-3-trichloromethyl-t,2,4-thiadi~ole ethyl 4-(methylthio)“m-tolyl isopropylphosphoramidate 3-(3,s dichlorophenyl)-N-(l-methylethyl)-ZCdioxo-limidazolidinecarboxamide zinc ion coordination product of manganese ethylenebisdithiocarbamate pentachloronitrobenzene bis(dimetbylthioc~bamoy1) disulfide
Chemical name
Table 2. Pesticide names, chemical compositions, and rates and numbers of applications at Ithaca
R. W. SMILEYand M. M. CRAVEN
358
as one drenching application in May; and (3) quintozene and ethazole, applied as nine foliar applications, every 7 days, in July and August. In addition, five treatments resembled commercial fungicide “programs”, consisting of two or three fungicides: (i) alternated so that any one material was applied at 42- or 63-day intervals; or (ii) alternated and also in combination with one or more of the repetitive treatments. An example would be the alternated use of ethyl thiophanate + thiram, chlorothalonil, and mancozeb, plus the repetitive mid-summer treatments of quintozene and ethazole. This program typifies a season-long commercial program designed to control activities of the phytopathogenic species of Drechslera, Sclerotinia, Fusarium, Rhizoctonia, and Pythium. All fungicide treatments were replicated three times in a randomized complete block design. Fusarium numbers in thatch + soil were estimated, as described for the Long Island plots, three times in each of 1976 and 1977: before resumption of the seasonal spray schedule in April; during mid-summer; and 2-3 weeks after the last seasonal application in September. Propagule numbers were converted to a proportion-of-control-treatment basis, and data were evaluated by a standard r-test (P = 0.05). Percentages of infected crowns were also determined in September 1977, as was the species’ composition of Fusarium (sensu Booth) in the infected crowns and in soil. During the field studies described above, Fusarium numbers increased greatly after only three iprodione fungicide applications had been made, but the increase did not continue as more applications were made. Consequently, two separate sets of replicated plots at the Mill River Club, which had been treated Table 3. Fusarium populations in thatch
Treatment (Code from Tables 1 & 2) A Bf Bd cs CP CH CY E F MC :
CYQ EtT MtM CHIA CHjMtM CH/MtM + F EtT/CH/Mc EtT/CH/Mc + Q + E * Proportions followed by t Propagules g-i (x 105) 2.6 (May), 2.3 (July) and 1.4 $ Nontreated controls had
RESULTS Quantitative
0.9 0.4* ND 0.5 0.8 0.7 1.0 ND ND 1.7; 0.9 1.0 0.7 ND ND ND ND ND ND ND 0.7
assessments
Applications of fungicides to Poa pratensis turfgrass caused both inhibitory and stimulatory effects on the numbers of fusaria in the plant crowns and in the soils. There were more crowns colonized by fusaria in the areas treated with fungicides ( x 1.s2.8) than in the nontreated control areas on fairways at the Mill River Club on Long Island. For turfgrasses treated with methyl thiophanate or with iprodione, crown colonization proportions ( x 2.s2.8) in 1977 were significantly higher than for the control turfgrass. Iprodione produced a similar stimulatory effect in the nontarget organism study at Ithaca, but benomyl, a near-equivalent of methyl thiophanate, reduced numbers of colonized crowns. The discrepancy between benomyl and methyl thiophanate at the Long Island and Ithaca locations in 1977 probably is the result of a high incidence of benomyl-tolerant fusaria at the Mill River Club, which does not occur
+ soil and proportions by fusaria at Ithaca
Soil populations 1976 June SePt 1.2 0.2* ND 1.0 0.8 0.8 0.9 ND ND 2.4* 0.9 1.0 0.7 ND ND ND ND ND ND ND 0.7
in 1976 with iprodione, at active ingredient rates of 6.6 kg ha- 1 (Smiley and Craven, 1977), and two sets of .replicated nontreated control plots were selected. In 1977, identical iprodione treatments were applied to one of the 1976-treated sets and to one of the nontreated control sets. This resulted in plots treated with iprodione in 1976 alone, in 1977 alone, in both 1976 and 1977, or left untreated. In late August 1977, Fusarium numbers in thatch + soil were estimated, and percentages of Fusarium-colonized crowns were determined.
(Prop. May 1.2 0.4’ 1.1 0.9 0.9 0.7 0.7 1.2 0.9 1.3 0.9 1.1 0.9 0.6 0.0’ 0.1* 0.6 0.3* 0.2* 0.4* 0.9
of Kentucky
bluegrass
of the control)t 1977 July Sept 1.0 0.18 0.9 0.7 0.9 1.0 1.3 1.8* 1.2 1.4* 1.0 1.0 0.8 0.8 0.2* 0.4* 0.9 0.4; 0.7 0.6 0.4;
crowns
colonized
Crowns colonizedj (Prop. of the controls) Sept 1977
0.8 0.4 1.5 0.7 0.8 0.8 0.9 1.5 1.0 1.5 1.1 1.1 1.1 0.8 0.2* 0.7 1.0 1.1 0.5 0.7 0.9
an asterisk differ (P = 0.05) from the nontreated control. in control plots in 1976 were 11.3 (June) and 1.5 (Sept), and (Sept). 10% of crowns infected by Fusarium. ND = not determined.
ND 0.4 ND 4.1’ ND 0.5 ND ND 2.2* 2.2’ 0.9 ND ND ND ND 0.7 ND ND ND ND ND
in 1977 were
Fusarium species in turfgrass at Ithaca (Smiley and Craven, 1979b). The nematicide fenamiphos and the fungicide cadmium succinate also caused significant increases in the frequency of Fusarium-colonized crowns at Ithaca. Fungicides did not greatly influence the numbers of fusuria in soils on the golf courses, with the exception of iprodione which caused significant increases ( x 2.1-3.0) after only three applications had been made. This fungicide, as well as ethazole, amplified Fusarium numbers at Ithaca. Significantly reduced numbers of fusaria occurred in the Ithaca plots treated with benomyl (fohar), ethyl thiophanate + thiram, and methyl thiophanate + maneb, all of which contain a benzimidazole-derivative ingredient. Insignificant reductions ( x 0.60.8) in soil fusaria also occurred in the Mill River Club plots treated with benomyl (1976) or with methyl thiophanate (1977). Fusarium species composed from 2 to 22% of all fungi isolated from the Ithaca plots (Smiley and Craven, 1978b), with averages of 5.1% for the controls and 5.7% for the 22 fungicide treatments. Ethazole (which is specific for pythiaceous fungi), quintozene, benomyl (drench), and ethyl thiophanate + thiram are characterized by Fusarlum-to-fungi ratios considerably higher than that of the control plot. As few as three high-dosage applications of iprodione to turfgrass invariably increased the number of fusaria in turfgrass crowns and in soil. But the effect was not greater in the long-term study at Ithaca, after a total of 25 applications, than in the single-season studies conducted on Long Island golf courses. On Mill River Club plots treated with iprodione in 1976 alone, in 1977 alone, in 1976 and 1977, or left untreated, the numbers of Fusarium propagules in soil during late August 1977 were 59, 48, 64 and 25 x 104g-‘, respectively, and the percentages of Fusarium-colonized crowns were 59, 38, 94, and 25%. Once again the stimulatory effect of iprodione was demonstrated, and the highest number and percentage occurred in plots sprayed for 2 yr. Qualitative assessments Identities of Fusarium species isolated from soils and from crowns were determined. Specific fungicides generally affected the species’ composition of plots more so than the total numbers of fusaria. The Fusarium species’ dominance in the nontreated control plot soil at Ithaca was distributed among F. graminearum, F. equiseti, F. acuminatum, F. oxysporum, and F. heterosporum (Table 4). Although species of the sections Arachnites, Arthrosporiella, and Sporotrichiella were not isolated from the control plots, representatives of Arthrosporiella were relatively common in plots treated with fungicides. Individual fungicides varied widely in their effect upon distributions of Fusarium species within the soil + thatch population, and none of the distributions in treated plots was identical to that in the control plot. Several groups of fungicides producing similar effects were separable. The cycloheximide and mancozeb treatments were reasonably similar to the controls, and it was shown earlier that these treatments also had little influence upon the numbers of fusaria in soil. Thiram, quintozene, and the alternated fungicide programs induced a slightly greater shift in species’ distributions than the previous group, and
359
several of these greatly reduced Fusarium numbers in soil. There are, thus, some treatments which reduce the numbers without significantly altering the species’ distribution. In contrast, about 88% of the isolates in captan-treated soil became uniformly distributed among six species, and the numbers of fusaria were only moderately reduced by this treatment. A fourth general group of fungicides caused the isolates of Fusarium to be concentrated within a small number of species. For example, about 75% of all isolates were within three species for the benomyl (foliar), iprodione, and cycloheximide + quintozene treatments, and within four species for methyl thiophanate + thiram, and the alternated fungicide treatments. Within these latter groups there were opposing trends in Fusarium populations, in that iprodione stimulated populations and most other treatments caused varying magnitudes of inhibition. Fungicides in the plot at Ithaca were thus shown to have influenced Fusarium population densities and species’ prevalence within a population in an independent manner. The most frequently isolated species from Kentucky bluegrass crowns at Ithaca were F. equiseti, F. acuminatum and F. robustum (Table 5). Although the number of identified Fusarium isolates (64) was low, Fusarium culmorum and F. tricinctum were not isolated from crowns, and they were infrequently isolated from soils. There was apparently no relationship between the relative prevalence of Fusarium species in coronal tissue with that in soil, but this relationship was not conclusive due to the small sample population obtained from plant crowns. Species’ dominance for the fusaria in soils at the Long Island golf courses (Table 4) was very different from that at Ithaca. The species’ composition during 1977 was similar for the two golf courses, with F. equiseti and F. merismoides dominating the population at Mill River (4588”/d) and at Nissequogue @O-90%). Species in the section Discolor were also frequently isolated at Mill River. At the latter golf course, F. merismoides and F. avenaceum were more prevalent in 1977 than in 1976, and F. oxysporum, F. sporotrichoides, and F. tricinctum were less prevalent. Although the 1976 and 1977 investigations were conducted on two different fairways, the sites were within 50m of each other, and the terrain, soils, and management were very similar. Weather differed between the 2yr, with 1977 being the wetter. At the Mill River Club, the treatment of turfgrass with iprodione or benomyl during 1976 had virtually no effect upon Fusarium species’ distributions (Table 4) although there were up to five-fold differences in the population densities. Fungicide influences upon the species’ composition were, however, evident during 1977, suggesting that environmental variables may affect the impact that fungicides have upon the soil mycoflora. During 1977, most of the species within the section Discolor were of F. robustum (sensu Gerlath, 1977) in the controls, F. graminearum in the methyl thiophanate treatment, and the two species were equally distributed in the iprodione treatment. The authors found that F. graminearum, F. heterosporum and F. robustum were difficult to differentiate. and therefore, when large numbers of fusaria are being identified, it may be desirable to combine these species, as is done in some other taxonomic systems
Q
Control A Bf Bd CS CP CH CY E F I MC
1thaca
66 53 29 97 38 55 55 74 91 61 99 74 78
Treatment No. (code from Tables 1 isolates examined and 2)
0 0
0 0 0 0 0 0 0 0 0 0 0
2
j
2 0 1 0 0 2 0 1 2 0 0 1
0
0 0 0 0 0 0 0 1 1 0 0 3 1
0 0 3 12 5 0 1 3 3 2 3 0
0
Arthrosporiella
: 0 0 2 2 0 0 1 0
0 0 0
23 I1 10 4 00600 18 2 3305 7 13 11 2 26 9 10 1 10 2 4720 16 8 9 10 7 0
13 5 3 5 2 0 0 0
2
9 13 7 9 9 4
0 0
S 12
Discolor
0 1 0 2 0 0 0
0 ;
0 0
11 18 18 12 13 15 15 9 16 14 10 11 10
Elegans
3 2 4 6 3 0 5 0 13 2 0 0 5
23 0 30 2 25 0 21 4 37 0 18 0 36 0 29 0 1 14 19 10 36 42 35 09 26 421
1 4 0 0 1 0 2 1 0 3
15 3 0 5 5 12 25
16 4
3 2 32
Liseola
15
Gibbosum
6 2 11 2 11 16 12 4 3 2 4 6 10
Martiella
8 0 0 0 0 0 0
2 0
0 0 0
P 2
x 9 0 0 0 0
0 1 0 0
0 0
5 ‘0 .c .I? t=
11 I 16 12 8 10 13 14 14 10 10 11
9
TotaI no. of species isolated
Kentucky bluegrass thatch + soil at Ithaca and on Long Island golf
Episphaeria
of Fusarium species and sections (sensu Booth) in fungicide-treated courses
Arachnites
Table 4. Percentage distribution
58
74
0 2 0 0 0
8
0 0 0 0 0
5 3 4
12 2 13
23
24 20 23
3
5 3 0
0
4 2 2
0
6 0
0 0 0 0
(Booth, 1971).
16060
12030
3 4 0
0
2 1 0
3
2 0
62 15 12
21
0 7 0 0
14 9 29 5 50 22 22
2 0 060400 2 8 5 24 0 2
: 7
0 0 0009020 0004060 0003040 0 0
0 0 0 0 0
0 2 0 0 0
: 0
: 1
0 0 0
0
0 1 0
0
0
0
0
0
:
0 0 0 0
0 0 0 0 5 0 0 0 0060340 4 :, 0 0042800
0 2 0 0 9 0 0 0
* F. robusturn (Gerlach, 1977) was separated from F. gramineurum
28 EtT 36 MtM IO CHJA 55 CH/MtM 34 CH/MtM + F 46 EtT/CH/Mc EtT/CH/Mc 58 +Q+E MillRiverClub-1976 100 Control 100 Bf 100 I MillRiverC~ub-197~ 46 Control 49 GA 60 I 39 Mt 52 Td NissequogueGolfClub-I977 66 Control 44 GA 110 I 68 Mt 54 Td
CYQ
T
0 0 0
0
0 0 0
0
2 0
0 0 0 0
30 78 10 24 41 46 53 45 49 17
3 0 0 0 0 2 0 2 0 5
4
I 2 2 3
; 0 4 4 3 2
5 18 7
8
8 17 24 25 12 1 6 21
0 0 0 0 0
0 0 8 0 0
2 3 1
34
31 26 17 16 31 37 39 36
0 0 0 0 0
0 0 0 0 0
0 0 0
3
1 3 5 0 8 12 10 0
44 27 43 40 65
36 10 45 21 42
40 35 49
14
9 0 17 3 15 22 3 4
0 0 0 0 0
8
:: 0
::
1
7
f 12 0
3 0 4 3
8 0
0 0
6 11 5
0 :: 8 2 0 0
0 0 2 17 0 2 7 0 4 0
12 14 13
4 2 0
0 0 1
0 1 1
8 5 7 6 5 5 6
0 0 0 0 0 0 0
10
0
0
4
9 9 10 7 12 13 8 9
0 0 5 0 0 0 0 6
0 0 0 0 0 0 0 0
8 9 11 9 4 1 0 17
b
?!
B 8 W’ VI 5‘
i 2. i
R. W.
362 Table
5. ~u~ri~~
SMILEY and
species
isolated from Kentucky Ithaca
Control F. F. F. F. F. F. F. F. F.
acuminatum avenaceum equiseti graminearum merjs~~des oxysporu~ robusturn sambucinum solani Total t No. of isolates
M. M. CRAVEN
1t 0 4 2 0 0 2
bluegrass
Treatment (code from Table Bf CH CS F 0 0 0 0 1 0 1
2 0 0 0 0 0
1
0
i
0 5
0 2
0 2
2 1 2 2 4 3 3 2 0 8
crowns
2) I
at
MtM
1 1
2
0
0
0
5 0 0 0 6 0 0 4
3 1 0 1 3 0 3 6
0’ 0
1 t!l 0 3
of each species.
(Snyder and Hansen, 1945). If this is done, then it is apparent that iprodione and be~imidazole fungicides, which had no effect upon species’ dominance in 1976, again did not alter the distribution of numbers within Discolor during 1977. In contrast, the experimental fungicides CGA 64251 and triadimefon greatly reduced percentages of these species (Table 4) without affecting the total population in soil; in both cases the species’ distribution was shifted from the section Discolor to the sections Ephisphaeria and Gibbosum. Single isolates of two species were recorded, and were not included in Table 4. At Ithaca the control treatment yielded one isolate of F. uentricosum, and the chlorothalonil-alternated-with-anilazine treatment yielded one isolate of F. aquaeductuum var. medium. DLSCUSSlON
Fusarium species were always present in turfgrasses, even after that ecosystem had been exposed to the severe selection pressure of up to six fungicides applied repeatedly for several years. Although fungicides generally either depressed the population or had little effect, several fungicides amplified the population of fusaria in turfgrass. The qualitative effects of fungicides on fusaria were also highly variable and were independent of the quantitative effects. Fusurium-to-total fungus ratios and Fusarium species’ compositions for these turfgrass studies were in general agreement with those previously reported for temperate or cold-temperate zone grassland or cereal soils. Fusaria represent 12-22% of all fungi in Canadian cereal-producing soils (Gordon, 1954), the most common species of which are F. oxysporum, F. equiseti, F. acuminatum, F. solani, and F. merismoides. Similar findings have been made in pasture soils of Iceland (Kommedahl and Siggeirsson, 1973), and in the turfgrasses in New York. Numbers of fusaria could be expected to increase in the presence of fungicides which are not inhibitory to Fusarium (Domsch, 1964), and this enrichment of fusaria could in turn lead to an increased incidence of Fusarium-caused diseases. The effect of quintozene and toxicants selective against pythiaceous fungi, such as ethazole, are particularly well-known for these effects. Our results corroborate this: Fusarium propagule numbers and Fusarium blight (Smiley et af.,
1978) were increased or unchanged by quintozene and ethazole. Other chemicals were major exceptions to this principle: mancozeb had little effect on the fusaria but greatly suppressed Fusarium blight; and iprodione always increased numbers of fusaria and completely prevented the disease’s occurrence on the Ithaca plot (Smiley et al., 1978). The iprodione fungicide is essentially nonfungitoxic toward fusaria (Smiley and Craven, 1979b), and thus, the mechanism whereby it controls this disease remains unknown. No apparent relationships existed between the species’ composition of Fusar~um in soil and that in coronal tissue in this study or in the cereal and pasture investigations discussed previously. The species frequently associated with crowns in our study have previously been isolated from roots (Booth, 1971: Domsch and Gams, 1972; Gordon, 1959; Kommedahl and Siggeirsson, 1973; Thornton, 1965). HOWever, F. merismoides, which occurred in crowns at Ithaca, is considered to be a soil inhabitant rather than a root inhabitant and occurs infrequently in root and coronal tissue (Kommedahl and Siggeirsson, 1973). Percentages of apparently healthy crowns which were colonized by fusaria ranged from 4 to 56”i,. Papendick and Cook (1974) have reported similar percentages of F. culwwrum-colonized crowns of wheat plants which remained healthy unless environmental stresses were exerted on the plant. In our study, fungicide treatments which controlled the disease (Smiley and Craven, 1977; Smiley er ~1.. 1978) often increased the proportions of colonized crowns. The proportions of colonized crowns were inversely related to Fusarium blight severity (unpublished data), which indicates that the role of F. culmorum. F. tricinctum, and other fusaria in the etiology of Fusarium blight should be reinvestigated. The presence of these ubiquitous fungi is a characteristic of even healthy turfgrasses, and their presence in or on plants may be unrelated to Fusarium blight.
Acknowledgements-We thank Drs P. E. Nelson and T. A. Toussoun, Fusarium Research Center, The Pennsylvania State University, University Park, PA for reviewing this manuscript, and for confirming our identifications of representative isolates of the Fusarium species examined in the course of this investigation.
Fusorium speck :s in turfgrass REFERENCES The Genus Fusarium. Commonwealth Mycol. Inst., Kew, Surrey, England. CLARK F. E. and PAUL E. A. (1970) The microflora of grassland. Advances in Agronomy 22, 375435. COLE H. JR, FORER L. B., NELSON P. E., BLOOM J. R. and JOD~N M. H. (1973) Stylet nematode genera and Fusarium species isolated from Pennsylvania turfgrass sod production fields. Plant Disease Reporter 57, 891-895. COUCH H. B. and BEDFORD E. R. (1966) Fusarium blight of turfgrasses. Phytopatho[ogy 56, 78 l-786. COUCH H. B. (1974) Diseases of Turfgrasses 2nd edn Kreiger, Huntington, New York. DOMSCH K. H. (1964) Soil fungicides. Annuul Review of Phyropurhology 2, 293-320. D~MSCH K. H. and GAMS W. (1972) Fungi in Agriculrural Soik. Wiley, New York. ENDS R. M. (1961) Turfgrass disease in southern California. Plant Disease Reporter 45, 869-873. GERLACH W. (1977) Fusarium robuswm spec. nov., der erreger einer stammftiule an Araucariu angustifolia (Bertol.) 0. Kuntze in Argentinien? Phytopathologische Zeirschrif 88, 29-37. GORDON W. L. (1954) The occurrence of Fusarium species in Canada. IV. Taxonomy and prevalence of Fusurium species in the soil of cereal plots. Canadian Journal of Botany 32, 622-629. GORDON W. L. (1959) The occurrence of Fusarium species in Canada. VI. Taxonomy and geographic distribution of Fusarium species on plants, insects, and fungi. Canadian Journul of Borany 37, 257-290. KOMMEDAHL T. and SIGGEIRSSON E. I. (1973) Prevalance of Fusarium species in roots and soil of grassland in Iceland. Research Inst. Nedri dis, Bulletin No. 14. Hveragerdi, Iceland. MES~~AENC. M. and CASSINI R. (1968) Recherches sur les Fusarioses IV. La systematique des Fusarium. An&es des dpiphyries 19, 387-454. NASH S. M. and SNYDER W. C. (1962) Quantitative estimaEDITH C. (1971)
363
tions by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopatholoyy 52, 567-572. ORPUR~ P. A. and CURTIS J. T. (19.57) Soil microfungi in relation to the prairie continuum in Wisconsin. Ecoloy) 38, 628-637. PAPENDICK R. I. and COOK R. J. (1974) Plant water stress and development of Fusarium root rot in wheat subjected to different cultural practices. Phytopatho/ogy 64, 358-363. SMILEY R. W. and CRAVEN M. M. (1977) Control of benzimidazole-tolerant Fuscrrium roseum on Kentucky bluegrass. Plum Disease Reporter. 61, 484-488. SMILEY R. W. and CRAVEN M. M. (1978) Fungicides in Kentucky bluegrass turf: Effects on thatch and pH. Agronomy Journal 70, 1013-1019. SMILEY R. W. and CRAVEN M. M. (1979a) Microflora of turfgrass treated with fungicides. Soil Biology & Biochemistry 11, 349-353. SMILEY R. W. and CRAVEN M. M. (1979b) In citro effects of Fusarium blight-controlling fungicides on pathogens of Poa prarensis. Soil Biology & Biochemistry II, 365370. SMILEY R. W., CRAVFN M. M. and THOMPXIN D. C. (1978) Helminthosporium leaf spot and Fusarium blight control on Kentucky bluegrass. Fungicide and Nematicide Tests 33, 145. SNYDER W. C. and HANSEN H. N. (1945) The species concept in Fusarium with reference to Discolor and other sections. American Journal of Botany 32, 657-666. THORNTON R. H. (1965) StudieS of fungi in pasture soils. I. Fungi associated with live roots. New Zealand Journal of Agriculrural Research 8, 417-449. TOUS~CIUNT. A. and NELSON P. A. (1976) A Pictoriul Guide to the Identijicution of Fusarium species, 2nd edn. Pennsylvania State Univ. Press, University Park. WAID J. S. (1957) Distribution of fungi within the decomposing tissues of ryegrass roots. Transactions of the British Mycological Society 40, 391-406. WARCUP J. H. (1951) The ecology of soil fungi. Transactions of rhe British Mycological Society 34, 376-399.
SPECIES IN SOIL, THATCH AND CROWNS OF POA PRATENSIS TURFGRASS TREATED WITH FUNGICIDES
FUSARIUM
R. W.
SMILEY
and M. M.
CRAVEN
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, U.S.A. (Accepted 10 March 1979) Summary-Little information exists concerning the effects of long-term fungicide programs on the qualitative and quantitative characteristics of fusaria in turfgrasses and in turfgrass soils. These effects were studied on Pea pratensis L. turfgrasses at three locations, using 14 fungicides, one nematicide, and five mixed fungicide programs. Some fungicides increased the numbers of fusaria in soil and thatch, some had no effect, and others greatly reduced the numbers. Changes in Fusarium species’ compositions occurred independently from the changes in propagule numbers. The proportion of Fusarrum-colonized turfgrass crowns was generally higher in fungicide-treated than in nontreated turfgrasses.
lNTRODUCTION
Fus~rium species are among the most abundant fungi in grassland soils, and they are invariably associated with the roots of Gramineae. The dominant fusaria in grasslands include F. oxysporum, F. solani, F. tri-
cinctum, and various species within the compositegroup F. roseum emend. Snyder & Hansen (Clark and Paul, 1970; Grpurt and Curtis, 1957; Warcup, 1951). In a survey of the fusaria in crowns and roots of apparently healthy Poa prutensis L. (Kentucky bluegrass) being produced for sod, Cole et al. (1973) isolated F. roseum and F. tr~ci~c~m from 100% and 37”/, respectively, of ah fields sampled. Kommedahl and Siggeirsson (1973) have also isolated fusaria from 69 of 71 fields surveyed in Iceland, with the F. roseumtypes being the species most frequently isolated from the roots and from the soil. The second most abundant species on roots, and third-ranked in soil, was F. oxysporum. Roots often also yielded F. tricinctum. Fusarium species are undoubtedly a very important component of grasstand ecosystems (Domsch and Gams, 19721, and their dominance over other fungi is greatest on senescing roots (Waid, 1957). Kentucky bluegrass is one of the most widely used turfgrasses in the United States. One of the important factors limiting its successful culture is a crowii rot named Fusarium blight (Couch and Bedford, 1966; Endo, 1961). Although the etiology of Fusarium blight is not understood, the incitants have been considered to be F. roseum f. sp. cereaiis (Cke.} Snyd. & Hans. “Culmorum” and F. rricinctum f. sp. pose (Pk.) Snyd. & Hans. (Couch, 1974). These and other fusaria are generally associated with senescing leaf tissues on diseased plants, and are also often isolated from decaying plant crowns. Fusarium blight is prevalent only on older stands of Kentucky bluegrass that are highly maintained as golf course fairways, home lawns, or commercial building landscapes. A characteristic of the intense management given to these areas is the routine application of pesticides. The influence of fungicides on the Fusarium species’ distribution in turfgrasses has
not been investigated. An understanding of this interaction is especially important on Fusarium blightprone turfgrass sites, for if a fungicide that is regularly applied to control another disease leads indirectly to an increase in the inoculum potential of pathogenic fusaria, that fungicide’s use could be counter-productive to subsequent Fusarium blight control measures. Knowledge about fungicide-Fusarium interactions is also basic to achieving an understanding of the etiology of Fusarium blight. We report the occurrence of Fusarium species on golf course fairways where Fusarium blight control investigations have been conducted (Smiley and Craven, 1977; Smiley et al., 1978), and on long-term plots where fungicides’ effects on nontarget soil organisms and on microbial processes are being studied (Smiley and Craven, 1978, 1979a). MATERIALS AND METHODS
Disease control studies on Long ls~~nd
Fungicides were evaluated during 1976 and 1977 on 12-yr-old Kentucky bluegrass fairways at the Mill River Club, Oyster Bay, NY. Plots were established on fairway areas that had been uniformly affected by Fusarium blight the previous year, and they were given the golf course’s complete fairway maintenance program, including applications of fungicides for control of other diseases. None of these fungicides appeared to alter the incidence of Fusarium blight. In 1977, another plot was established at the Nissequogue Golf Club, St. James, NY, where Fusarium blight had occurred on a IO-yr-old Kentucky bluegrass fairway in 1976. The Mill River fairways are routinely watered with automatic irrigation systems, whereas those at Nissequogue are not. Experimental designs were randomized complete blocks with four replications per treatment. Applications of fungicides began about 20 May each year, and all golf-course resuhs reported here are from treatments in which fungicides were drenched into the thatch or soil with supplemental sprinkler irrigation
R. W. SMILEY and
356 Table
1. Pesticide
names*
M. M. CRAVFN
and application
rates on Long
Island
golf courses
Applications
(kg a.i. ha-
1976 Treatment code BF
I Mt Td GA * Chemical
Trade Common
name
benomyl iprodione methyl thiophanate triadimefon (none) names
are the same
1977
(?
June 7
June 21
Aug. 2
May 24
June 12
July 11
Tersan 1991 (50) Chipco 26019 (50) Spot Kleen (70) Bayleton (SO) CGA 64251 (21.5)
11.2 11.2 0 0 0
11.2 11.2 0 0 0
6.6 6.6 0 0 0
0 11.2 11.2 3.0 2.5
0 11.2 11.2 3.0 2.5
0 11.2 11.2 3.0 2.5
as those
name
’)
listed
in Table
2, except for triadimefon, which is Names for CGA 64251 are
1-(4-chlorophenoxy)-3,3-dimethyl-1-( lH-1,2,4-triazol-1-yI)-2-butanone. confidential.
and immediately after applications. Details of applications, plot areas, and additional treatments are given by Smiley and Craven (1977) and by Smiley ef al. (1978). Treatments reported here are summarized in Table 1. During the last week in August each year, seven cores, of 2.5 cm dia x 2 cm, with thatch intact, were collected from random locations within each treatment area. Cores were placed in plastic bags and were refrigerated during transport to the laboratory. The cores were then chopped, mixed thoroughly, and log of each thatch + soil mixture were suspended in 95 ml 0.1% water agar in a 200 ml milk dilution bottle containing 250 (3 mm dia) glass beads. A portion of the remaining thatch + soil was dried to permit adjustment of the data to an oven-dry soil basis. The suspensions were then placed horizontally in a reciprocating shaker and shaken vigorously for 10 min at 200 cycles min-‘. The suspension was diluted in a lo-fold dilution series in O.l:/, water agar, and 1 ml of an appropriate dilution was dispensed aseptically onto plates of peptone-PCNB-agar medium (Nash and Snyder, 1962). After incubation of the plates for 4 ~7 days at 25”C, the fusaria were counted and propagule numbers were adjusted to an oven-dry soil basis. Fusarium isolates were then transferred from the Nash and Snyder medium to plates of potato-dextrose-agar (PDA) and of carnation-leaf-agar (CLA) (Toussoun and Nelson, 1976). Plates were incubated together at about 25°C under fluorescent lights for 410 days. Fusarium species were identified by observing macroscopic characteristics of PDA colonies and microscopic details from colonies growing on CLA; the taxonomic system of Booth (1971) was used throughout this study. Many representative isolates were submitted to the Fusarium Research Center, The Pennsylvania State University, for confirmed diagnoses by Drs T. A. Toussoun and P. E. Nelson. The taxonomic system of Booth does not recognize F. robustum as a species distinct from F. yraminearum. The separation was made in our study in view of its prevalence in the turfgrasses of New York State. Gerlach (1977) described similar isolates as F. robusrum and later, utilizing isolates from the Mill River Club, which were submitted for comparison by Dr T. A. Toussoun, showed that these were also similar to F. robustum. Personal communications with Dr P. E. Nelson indicate that isolates similar to F. robusduring
turn were also shown by Dr Lester Burgess to be common in some pasture soils in Australia, and that they were considered by Dr C. M. Messiaen to be a “soiltype of F. graminearum” (Messiaen and Cassini, 1968). The species designation F. robustum was, therefore, selected in our study as a supplement to the taxonomic system of Booth (1971). Identifications in 1976 were made for 100 randomly-selected isolates per fungicide treatment, and in 1977, to exclude the possibility for non-randomlybiased selections of Fusarium isolates, every isolate from the Nash and Snyder medium was identified; the numbers of identifications per treatment ranged from 39 to 110 (average of 59). Fusarium species were also isolated from the crowns of all individual tillers present in the 1976 and 1977 core samples. Foliage and roots were trimmed from coronal tissue, and the crowns were then placed in cheesecloth bags and washed under rapidly-running tapwater for 223 h. Crowns were blotted dry on clean paper towels, soaked in a 1 :l mixture of sodium hypochlorite (5.25%) and ethanol (95%) for 1 min. rinsed in distilled water, and then spaced individually on acidified PDA medium (pH 5.0). Numbers of inspected crowns ranged from 25 to 35/treatment in 1976 and from 65 to 95 in 1977. Crowns colonized by Fusarium roseum emend Synd. & Hans. were enumerated but identified by cultivar name. Nontarget
organism
study ut lthuca
A 2-yr-old Kentucky bluegrass turf was installed as sod in 1975 at Cornell University. Repeated applications of fungicides were then made to replicated plots (1 x 5 m) during 1975, 1976, and 1977. Details of the plots’ management, the soil, and the fungicide application equipment have been described by Smiley and Craven (1978). The nonirrigated turf was maintained at a height of 4.4 cm, and clippings were removed during mowing. Fourteen fungicides and one nematicide (Table 2) were selected as representatives of those likely to be used commercially. Foliar spray treatments were made nine times annually, every 21 days, from April until September. Exceptions to this schedule included: (1) benomyl, applied as nine foliar applications for one treatment, and as two drenching applications in June for another treatment: (2) fenamiphos applied
Actidione RZ (0.75 + 75)
Bromosan (17 + 50)
MF 598 (15 + 60)
quintozene (or PCNB) thiram cycloheximide + quintozene
ethyl thiophanate + thiram
methiophanate + maneb
Q
EtT
MtM
(as above; alternated) (as above; alternated) (as above; CH & MtM alternated, plus F) (as above; alternated) above; EtT, CH, & MC alternated, plus Q & E)
Terraclor (75) Tersan 75 (75)
* Drenching applications. All other applications were foliar.
CH/A CH/MtM CH/MtM + F EtT/CH/Mc EtT/CH/Mc + Q + E (as
CYQ
T
Dithane M-45 (80)
mancozeb
MC
Dyrene (50) Tersan 1991 (SO) Tersan 1991 (50) Cadminate (60) Captan (SO) Daconil 2787 (75) Actidione TGF (2.1) Koban (65) Nemacur (15) Chipco 26019 (50)
Trade name (% a.i.)
anilazine benomyl benomyl cadmium succinate captan chlorotbatonit cycloheximide ethazole fenamiphos iprodione
Common name
A Bf Bd CS CP CH CY E F I
Treatment code
514 514 514 + 1 3/3/3 31313 +9+9
9
9
9
(as (as (as (as (as
above) above) above) above) above)
2.3 + 12.2
3.1 + 9.2
0.1 + 1.7
6.9 9.2
9.8
9 9 9
2: 3:1
9 9 2* 9 9 9 9 9 1* 9
7.7 3.1 12.2 3.7 7.7 9.2 0.1
Annual applications Number kg ha-’ appl-’
dimethyl 4,4-a-phenylenebis[3-thioallophanatelplus manganese ethylenebisdithiocarbamate -
diethyl 4,4-u-phenylenebis[3-thioallophanatelplus bis(dimethylthiocarbamoyl)disulfide
~-[2-(3,5-dimethyl-2-oxocyclohexyl~2-hydroxyethyl] glutarimide plus pentachloronitrobenzene
2,4-dichloro-6-(o-chloroanilino)-s-triazine methyl I-(butylcarbamoylf-2-benzimidazolecarbamate methyl I-(butylcarbamoyl)-2-benzimidazolecarbamate cadmium succinate N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide tetrachloroisophthaionitriie ~-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide 5-ethoxy-3-trichloromethyl-t,2,4-thiadi~ole ethyl 4-(methylthio)“m-tolyl isopropylphosphoramidate 3-(3,s dichlorophenyl)-N-(l-methylethyl)-ZCdioxo-limidazolidinecarboxamide zinc ion coordination product of manganese ethylenebisdithiocarbamate pentachloronitrobenzene bis(dimetbylthioc~bamoy1) disulfide
Chemical name
Table 2. Pesticide names, chemical compositions, and rates and numbers of applications at Ithaca
R. W. SMILEYand M. M. CRAVEN
358
as one drenching application in May; and (3) quintozene and ethazole, applied as nine foliar applications, every 7 days, in July and August. In addition, five treatments resembled commercial fungicide “programs”, consisting of two or three fungicides: (i) alternated so that any one material was applied at 42- or 63-day intervals; or (ii) alternated and also in combination with one or more of the repetitive treatments. An example would be the alternated use of ethyl thiophanate + thiram, chlorothalonil, and mancozeb, plus the repetitive mid-summer treatments of quintozene and ethazole. This program typifies a season-long commercial program designed to control activities of the phytopathogenic species of Drechslera, Sclerotinia, Fusarium, Rhizoctonia, and Pythium. All fungicide treatments were replicated three times in a randomized complete block design. Fusarium numbers in thatch + soil were estimated, as described for the Long Island plots, three times in each of 1976 and 1977: before resumption of the seasonal spray schedule in April; during mid-summer; and 2-3 weeks after the last seasonal application in September. Propagule numbers were converted to a proportion-of-control-treatment basis, and data were evaluated by a standard r-test (P = 0.05). Percentages of infected crowns were also determined in September 1977, as was the species’ composition of Fusarium (sensu Booth) in the infected crowns and in soil. During the field studies described above, Fusarium numbers increased greatly after only three iprodione fungicide applications had been made, but the increase did not continue as more applications were made. Consequently, two separate sets of replicated plots at the Mill River Club, which had been treated Table 3. Fusarium populations in thatch
Treatment (Code from Tables 1 & 2) A Bf Bd cs CP CH CY E F MC :
CYQ EtT MtM CHIA CHjMtM CH/MtM + F EtT/CH/Mc EtT/CH/Mc + Q + E * Proportions followed by t Propagules g-i (x 105) 2.6 (May), 2.3 (July) and 1.4 $ Nontreated controls had
RESULTS Quantitative
0.9 0.4* ND 0.5 0.8 0.7 1.0 ND ND 1.7; 0.9 1.0 0.7 ND ND ND ND ND ND ND 0.7
assessments
Applications of fungicides to Poa pratensis turfgrass caused both inhibitory and stimulatory effects on the numbers of fusaria in the plant crowns and in the soils. There were more crowns colonized by fusaria in the areas treated with fungicides ( x 1.s2.8) than in the nontreated control areas on fairways at the Mill River Club on Long Island. For turfgrasses treated with methyl thiophanate or with iprodione, crown colonization proportions ( x 2.s2.8) in 1977 were significantly higher than for the control turfgrass. Iprodione produced a similar stimulatory effect in the nontarget organism study at Ithaca, but benomyl, a near-equivalent of methyl thiophanate, reduced numbers of colonized crowns. The discrepancy between benomyl and methyl thiophanate at the Long Island and Ithaca locations in 1977 probably is the result of a high incidence of benomyl-tolerant fusaria at the Mill River Club, which does not occur
+ soil and proportions by fusaria at Ithaca
Soil populations 1976 June SePt 1.2 0.2* ND 1.0 0.8 0.8 0.9 ND ND 2.4* 0.9 1.0 0.7 ND ND ND ND ND ND ND 0.7
in 1976 with iprodione, at active ingredient rates of 6.6 kg ha- 1 (Smiley and Craven, 1977), and two sets of .replicated nontreated control plots were selected. In 1977, identical iprodione treatments were applied to one of the 1976-treated sets and to one of the nontreated control sets. This resulted in plots treated with iprodione in 1976 alone, in 1977 alone, in both 1976 and 1977, or left untreated. In late August 1977, Fusarium numbers in thatch + soil were estimated, and percentages of Fusarium-colonized crowns were determined.
(Prop. May 1.2 0.4’ 1.1 0.9 0.9 0.7 0.7 1.2 0.9 1.3 0.9 1.1 0.9 0.6 0.0’ 0.1* 0.6 0.3* 0.2* 0.4* 0.9
of Kentucky
bluegrass
of the control)t 1977 July Sept 1.0 0.18 0.9 0.7 0.9 1.0 1.3 1.8* 1.2 1.4* 1.0 1.0 0.8 0.8 0.2* 0.4* 0.9 0.4; 0.7 0.6 0.4;
crowns
colonized
Crowns colonizedj (Prop. of the controls) Sept 1977
0.8 0.4 1.5 0.7 0.8 0.8 0.9 1.5 1.0 1.5 1.1 1.1 1.1 0.8 0.2* 0.7 1.0 1.1 0.5 0.7 0.9
an asterisk differ (P = 0.05) from the nontreated control. in control plots in 1976 were 11.3 (June) and 1.5 (Sept), and (Sept). 10% of crowns infected by Fusarium. ND = not determined.
ND 0.4 ND 4.1’ ND 0.5 ND ND 2.2* 2.2’ 0.9 ND ND ND ND 0.7 ND ND ND ND ND
in 1977 were
Fusarium species in turfgrass at Ithaca (Smiley and Craven, 1979b). The nematicide fenamiphos and the fungicide cadmium succinate also caused significant increases in the frequency of Fusarium-colonized crowns at Ithaca. Fungicides did not greatly influence the numbers of fusuria in soils on the golf courses, with the exception of iprodione which caused significant increases ( x 2.1-3.0) after only three applications had been made. This fungicide, as well as ethazole, amplified Fusarium numbers at Ithaca. Significantly reduced numbers of fusaria occurred in the Ithaca plots treated with benomyl (fohar), ethyl thiophanate + thiram, and methyl thiophanate + maneb, all of which contain a benzimidazole-derivative ingredient. Insignificant reductions ( x 0.60.8) in soil fusaria also occurred in the Mill River Club plots treated with benomyl (1976) or with methyl thiophanate (1977). Fusarium species composed from 2 to 22% of all fungi isolated from the Ithaca plots (Smiley and Craven, 1978b), with averages of 5.1% for the controls and 5.7% for the 22 fungicide treatments. Ethazole (which is specific for pythiaceous fungi), quintozene, benomyl (drench), and ethyl thiophanate + thiram are characterized by Fusarlum-to-fungi ratios considerably higher than that of the control plot. As few as three high-dosage applications of iprodione to turfgrass invariably increased the number of fusaria in turfgrass crowns and in soil. But the effect was not greater in the long-term study at Ithaca, after a total of 25 applications, than in the single-season studies conducted on Long Island golf courses. On Mill River Club plots treated with iprodione in 1976 alone, in 1977 alone, in 1976 and 1977, or left untreated, the numbers of Fusarium propagules in soil during late August 1977 were 59, 48, 64 and 25 x 104g-‘, respectively, and the percentages of Fusarium-colonized crowns were 59, 38, 94, and 25%. Once again the stimulatory effect of iprodione was demonstrated, and the highest number and percentage occurred in plots sprayed for 2 yr. Qualitative assessments Identities of Fusarium species isolated from soils and from crowns were determined. Specific fungicides generally affected the species’ composition of plots more so than the total numbers of fusaria. The Fusarium species’ dominance in the nontreated control plot soil at Ithaca was distributed among F. graminearum, F. equiseti, F. acuminatum, F. oxysporum, and F. heterosporum (Table 4). Although species of the sections Arachnites, Arthrosporiella, and Sporotrichiella were not isolated from the control plots, representatives of Arthrosporiella were relatively common in plots treated with fungicides. Individual fungicides varied widely in their effect upon distributions of Fusarium species within the soil + thatch population, and none of the distributions in treated plots was identical to that in the control plot. Several groups of fungicides producing similar effects were separable. The cycloheximide and mancozeb treatments were reasonably similar to the controls, and it was shown earlier that these treatments also had little influence upon the numbers of fusaria in soil. Thiram, quintozene, and the alternated fungicide programs induced a slightly greater shift in species’ distributions than the previous group, and
359
several of these greatly reduced Fusarium numbers in soil. There are, thus, some treatments which reduce the numbers without significantly altering the species’ distribution. In contrast, about 88% of the isolates in captan-treated soil became uniformly distributed among six species, and the numbers of fusaria were only moderately reduced by this treatment. A fourth general group of fungicides caused the isolates of Fusarium to be concentrated within a small number of species. For example, about 75% of all isolates were within three species for the benomyl (foliar), iprodione, and cycloheximide + quintozene treatments, and within four species for methyl thiophanate + thiram, and the alternated fungicide treatments. Within these latter groups there were opposing trends in Fusarium populations, in that iprodione stimulated populations and most other treatments caused varying magnitudes of inhibition. Fungicides in the plot at Ithaca were thus shown to have influenced Fusarium population densities and species’ prevalence within a population in an independent manner. The most frequently isolated species from Kentucky bluegrass crowns at Ithaca were F. equiseti, F. acuminatum and F. robustum (Table 5). Although the number of identified Fusarium isolates (64) was low, Fusarium culmorum and F. tricinctum were not isolated from crowns, and they were infrequently isolated from soils. There was apparently no relationship between the relative prevalence of Fusarium species in coronal tissue with that in soil, but this relationship was not conclusive due to the small sample population obtained from plant crowns. Species’ dominance for the fusaria in soils at the Long Island golf courses (Table 4) was very different from that at Ithaca. The species’ composition during 1977 was similar for the two golf courses, with F. equiseti and F. merismoides dominating the population at Mill River (4588”/d) and at Nissequogue @O-90%). Species in the section Discolor were also frequently isolated at Mill River. At the latter golf course, F. merismoides and F. avenaceum were more prevalent in 1977 than in 1976, and F. oxysporum, F. sporotrichoides, and F. tricinctum were less prevalent. Although the 1976 and 1977 investigations were conducted on two different fairways, the sites were within 50m of each other, and the terrain, soils, and management were very similar. Weather differed between the 2yr, with 1977 being the wetter. At the Mill River Club, the treatment of turfgrass with iprodione or benomyl during 1976 had virtually no effect upon Fusarium species’ distributions (Table 4) although there were up to five-fold differences in the population densities. Fungicide influences upon the species’ composition were, however, evident during 1977, suggesting that environmental variables may affect the impact that fungicides have upon the soil mycoflora. During 1977, most of the species within the section Discolor were of F. robustum (sensu Gerlath, 1977) in the controls, F. graminearum in the methyl thiophanate treatment, and the two species were equally distributed in the iprodione treatment. The authors found that F. graminearum, F. heterosporum and F. robustum were difficult to differentiate. and therefore, when large numbers of fusaria are being identified, it may be desirable to combine these species, as is done in some other taxonomic systems
Q
Control A Bf Bd CS CP CH CY E F I MC
1thaca
66 53 29 97 38 55 55 74 91 61 99 74 78
Treatment No. (code from Tables 1 isolates examined and 2)
0 0
0 0 0 0 0 0 0 0 0 0 0
2
j
2 0 1 0 0 2 0 1 2 0 0 1
0
0 0 0 0 0 0 0 1 1 0 0 3 1
0 0 3 12 5 0 1 3 3 2 3 0
0
Arthrosporiella
: 0 0 2 2 0 0 1 0
0 0 0
23 I1 10 4 00600 18 2 3305 7 13 11 2 26 9 10 1 10 2 4720 16 8 9 10 7 0
13 5 3 5 2 0 0 0
2
9 13 7 9 9 4
0 0
S 12
Discolor
0 1 0 2 0 0 0
0 ;
0 0
11 18 18 12 13 15 15 9 16 14 10 11 10
Elegans
3 2 4 6 3 0 5 0 13 2 0 0 5
23 0 30 2 25 0 21 4 37 0 18 0 36 0 29 0 1 14 19 10 36 42 35 09 26 421
1 4 0 0 1 0 2 1 0 3
15 3 0 5 5 12 25
16 4
3 2 32
Liseola
15
Gibbosum
6 2 11 2 11 16 12 4 3 2 4 6 10
Martiella
8 0 0 0 0 0 0
2 0
0 0 0
P 2
x 9 0 0 0 0
0 1 0 0
0 0
5 ‘0 .c .I? t=
11 I 16 12 8 10 13 14 14 10 10 11
9
TotaI no. of species isolated
Kentucky bluegrass thatch + soil at Ithaca and on Long Island golf
Episphaeria
of Fusarium species and sections (sensu Booth) in fungicide-treated courses
Arachnites
Table 4. Percentage distribution
58
74
0 2 0 0 0
8
0 0 0 0 0
5 3 4
12 2 13
23
24 20 23
3
5 3 0
0
4 2 2
0
6 0
0 0 0 0
(Booth, 1971).
16060
12030
3 4 0
0
2 1 0
3
2 0
62 15 12
21
0 7 0 0
14 9 29 5 50 22 22
2 0 060400 2 8 5 24 0 2
: 7
0 0 0009020 0004060 0003040 0 0
0 0 0 0 0
0 2 0 0 0
: 0
: 1
0 0 0
0
0 1 0
0
0
0
0
0
:
0 0 0 0
0 0 0 0 5 0 0 0 0060340 4 :, 0 0042800
0 2 0 0 9 0 0 0
* F. robusturn (Gerlach, 1977) was separated from F. gramineurum
28 EtT 36 MtM IO CHJA 55 CH/MtM 34 CH/MtM + F 46 EtT/CH/Mc EtT/CH/Mc 58 +Q+E MillRiverClub-1976 100 Control 100 Bf 100 I MillRiverC~ub-197~ 46 Control 49 GA 60 I 39 Mt 52 Td NissequogueGolfClub-I977 66 Control 44 GA 110 I 68 Mt 54 Td
CYQ
T
0 0 0
0
0 0 0
0
2 0
0 0 0 0
30 78 10 24 41 46 53 45 49 17
3 0 0 0 0 2 0 2 0 5
4
I 2 2 3
; 0 4 4 3 2
5 18 7
8
8 17 24 25 12 1 6 21
0 0 0 0 0
0 0 8 0 0
2 3 1
34
31 26 17 16 31 37 39 36
0 0 0 0 0
0 0 0 0 0
0 0 0
3
1 3 5 0 8 12 10 0
44 27 43 40 65
36 10 45 21 42
40 35 49
14
9 0 17 3 15 22 3 4
0 0 0 0 0
8
:: 0
::
1
7
f 12 0
3 0 4 3
8 0
0 0
6 11 5
0 :: 8 2 0 0
0 0 2 17 0 2 7 0 4 0
12 14 13
4 2 0
0 0 1
0 1 1
8 5 7 6 5 5 6
0 0 0 0 0 0 0
10
0
0
4
9 9 10 7 12 13 8 9
0 0 5 0 0 0 0 6
0 0 0 0 0 0 0 0
8 9 11 9 4 1 0 17
b
?!
B 8 W’ VI 5‘
i 2. i
R. W.
362 Table
5. ~u~ri~~
SMILEY and
species
isolated from Kentucky Ithaca
Control F. F. F. F. F. F. F. F. F.
acuminatum avenaceum equiseti graminearum merjs~~des oxysporu~ robusturn sambucinum solani Total t No. of isolates
M. M. CRAVEN
1t 0 4 2 0 0 2
bluegrass
Treatment (code from Table Bf CH CS F 0 0 0 0 1 0 1
2 0 0 0 0 0
1
0
i
0 5
0 2
0 2
2 1 2 2 4 3 3 2 0 8
crowns
2) I
at
MtM
1 1
2
0
0
0
5 0 0 0 6 0 0 4
3 1 0 1 3 0 3 6
0’ 0
1 t!l 0 3
of each species.
(Snyder and Hansen, 1945). If this is done, then it is apparent that iprodione and be~imidazole fungicides, which had no effect upon species’ dominance in 1976, again did not alter the distribution of numbers within Discolor during 1977. In contrast, the experimental fungicides CGA 64251 and triadimefon greatly reduced percentages of these species (Table 4) without affecting the total population in soil; in both cases the species’ distribution was shifted from the section Discolor to the sections Ephisphaeria and Gibbosum. Single isolates of two species were recorded, and were not included in Table 4. At Ithaca the control treatment yielded one isolate of F. uentricosum, and the chlorothalonil-alternated-with-anilazine treatment yielded one isolate of F. aquaeductuum var. medium. DLSCUSSlON
Fusarium species were always present in turfgrasses, even after that ecosystem had been exposed to the severe selection pressure of up to six fungicides applied repeatedly for several years. Although fungicides generally either depressed the population or had little effect, several fungicides amplified the population of fusaria in turfgrass. The qualitative effects of fungicides on fusaria were also highly variable and were independent of the quantitative effects. Fusurium-to-total fungus ratios and Fusarium species’ compositions for these turfgrass studies were in general agreement with those previously reported for temperate or cold-temperate zone grassland or cereal soils. Fusaria represent 12-22% of all fungi in Canadian cereal-producing soils (Gordon, 1954), the most common species of which are F. oxysporum, F. equiseti, F. acuminatum, F. solani, and F. merismoides. Similar findings have been made in pasture soils of Iceland (Kommedahl and Siggeirsson, 1973), and in the turfgrasses in New York. Numbers of fusaria could be expected to increase in the presence of fungicides which are not inhibitory to Fusarium (Domsch, 1964), and this enrichment of fusaria could in turn lead to an increased incidence of Fusarium-caused diseases. The effect of quintozene and toxicants selective against pythiaceous fungi, such as ethazole, are particularly well-known for these effects. Our results corroborate this: Fusarium propagule numbers and Fusarium blight (Smiley et af.,
1978) were increased or unchanged by quintozene and ethazole. Other chemicals were major exceptions to this principle: mancozeb had little effect on the fusaria but greatly suppressed Fusarium blight; and iprodione always increased numbers of fusaria and completely prevented the disease’s occurrence on the Ithaca plot (Smiley et al., 1978). The iprodione fungicide is essentially nonfungitoxic toward fusaria (Smiley and Craven, 1979b), and thus, the mechanism whereby it controls this disease remains unknown. No apparent relationships existed between the species’ composition of Fusar~um in soil and that in coronal tissue in this study or in the cereal and pasture investigations discussed previously. The species frequently associated with crowns in our study have previously been isolated from roots (Booth, 1971: Domsch and Gams, 1972; Gordon, 1959; Kommedahl and Siggeirsson, 1973; Thornton, 1965). HOWever, F. merismoides, which occurred in crowns at Ithaca, is considered to be a soil inhabitant rather than a root inhabitant and occurs infrequently in root and coronal tissue (Kommedahl and Siggeirsson, 1973). Percentages of apparently healthy crowns which were colonized by fusaria ranged from 4 to 56”i,. Papendick and Cook (1974) have reported similar percentages of F. culwwrum-colonized crowns of wheat plants which remained healthy unless environmental stresses were exerted on the plant. In our study, fungicide treatments which controlled the disease (Smiley and Craven, 1977; Smiley er ~1.. 1978) often increased the proportions of colonized crowns. The proportions of colonized crowns were inversely related to Fusarium blight severity (unpublished data), which indicates that the role of F. culmorum. F. tricinctum, and other fusaria in the etiology of Fusarium blight should be reinvestigated. The presence of these ubiquitous fungi is a characteristic of even healthy turfgrasses, and their presence in or on plants may be unrelated to Fusarium blight.
Acknowledgements-We thank Drs P. E. Nelson and T. A. Toussoun, Fusarium Research Center, The Pennsylvania State University, University Park, PA for reviewing this manuscript, and for confirming our identifications of representative isolates of the Fusarium species examined in the course of this investigation.
Fusorium speck :s in turfgrass REFERENCES The Genus Fusarium. Commonwealth Mycol. Inst., Kew, Surrey, England. CLARK F. E. and PAUL E. A. (1970) The microflora of grassland. Advances in Agronomy 22, 375435. COLE H. JR, FORER L. B., NELSON P. E., BLOOM J. R. and JOD~N M. H. (1973) Stylet nematode genera and Fusarium species isolated from Pennsylvania turfgrass sod production fields. Plant Disease Reporter 57, 891-895. COUCH H. B. and BEDFORD E. R. (1966) Fusarium blight of turfgrasses. Phytopatho[ogy 56, 78 l-786. COUCH H. B. (1974) Diseases of Turfgrasses 2nd edn Kreiger, Huntington, New York. DOMSCH K. H. (1964) Soil fungicides. Annuul Review of Phyropurhology 2, 293-320. D~MSCH K. H. and GAMS W. (1972) Fungi in Agriculrural Soik. Wiley, New York. ENDS R. M. (1961) Turfgrass disease in southern California. Plant Disease Reporter 45, 869-873. GERLACH W. (1977) Fusarium robuswm spec. nov., der erreger einer stammftiule an Araucariu angustifolia (Bertol.) 0. Kuntze in Argentinien? Phytopathologische Zeirschrif 88, 29-37. GORDON W. L. (1954) The occurrence of Fusarium species in Canada. IV. Taxonomy and prevalence of Fusurium species in the soil of cereal plots. Canadian Journal of Botany 32, 622-629. GORDON W. L. (1959) The occurrence of Fusarium species in Canada. VI. Taxonomy and geographic distribution of Fusarium species on plants, insects, and fungi. Canadian Journul of Borany 37, 257-290. KOMMEDAHL T. and SIGGEIRSSON E. I. (1973) Prevalance of Fusarium species in roots and soil of grassland in Iceland. Research Inst. Nedri dis, Bulletin No. 14. Hveragerdi, Iceland. MES~~AENC. M. and CASSINI R. (1968) Recherches sur les Fusarioses IV. La systematique des Fusarium. An&es des dpiphyries 19, 387-454. NASH S. M. and SNYDER W. C. (1962) Quantitative estimaEDITH C. (1971)
363
tions by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopatholoyy 52, 567-572. ORPUR~ P. A. and CURTIS J. T. (19.57) Soil microfungi in relation to the prairie continuum in Wisconsin. Ecoloy) 38, 628-637. PAPENDICK R. I. and COOK R. J. (1974) Plant water stress and development of Fusarium root rot in wheat subjected to different cultural practices. Phytopatho/ogy 64, 358-363. SMILEY R. W. and CRAVEN M. M. (1977) Control of benzimidazole-tolerant Fuscrrium roseum on Kentucky bluegrass. Plum Disease Reporter. 61, 484-488. SMILEY R. W. and CRAVEN M. M. (1978) Fungicides in Kentucky bluegrass turf: Effects on thatch and pH. Agronomy Journal 70, 1013-1019. SMILEY R. W. and CRAVEN M. M. (1979a) Microflora of turfgrass treated with fungicides. Soil Biology & Biochemistry 11, 349-353. SMILEY R. W. and CRAVEN M. M. (1979b) In citro effects of Fusarium blight-controlling fungicides on pathogens of Poa prarensis. Soil Biology & Biochemistry II, 365370. SMILEY R. W., CRAVFN M. M. and THOMPXIN D. C. (1978) Helminthosporium leaf spot and Fusarium blight control on Kentucky bluegrass. Fungicide and Nematicide Tests 33, 145. SNYDER W. C. and HANSEN H. N. (1945) The species concept in Fusarium with reference to Discolor and other sections. American Journal of Botany 32, 657-666. THORNTON R. H. (1965) StudieS of fungi in pasture soils. I. Fungi associated with live roots. New Zealand Journal of Agriculrural Research 8, 417-449. TOUS~CIUNT. A. and NELSON P. A. (1976) A Pictoriul Guide to the Identijicution of Fusarium species, 2nd edn. Pennsylvania State Univ. Press, University Park. WAID J. S. (1957) Distribution of fungi within the decomposing tissues of ryegrass roots. Transactions of the British Mycological Society 40, 391-406. WARCUP J. H. (1951) The ecology of soil fungi. Transactions of rhe British Mycological Society 34, 376-399.