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Isolation of Fusarium species from common broad-leaved weeds and their pathogenicity to winter wheat
776
Mycol. Res. 98 (7): 7 7 6 7 8 0 (1994) Printed in Great Brifain
Isolation of Fusarium species from common broad-leaved weeds and their pathogenicity to winter wheat
P. J E N K I N S O N A N D D. W. PARRY Crop and Environment Research Centre, Harper Adams Agricultural College, Newport, Shropshire, TFlO 8NB. U.K.
Isolations were made from the stem-bases of 1346 plants representing 15 weed species collected from three sites of fallow land; one following a crop of potatoes and two following wheat crops. Two hundred and twenty-six Fwarium isolates were obtained from 14 of the 15 weed species of which 114 (50.4%) were F. avenaceum, 88 (38.9%) were F. culmorum, 16 (7.1%)were F. poae, 6 (2.5%) were F. sambucinum and 3 (1.3%) were F. graminearum. No obvious symptom of Fusarium infection was observed on any of the plants sampled. Seventy-five of 77 Fusarium isolates (33 F. avenaceum, 27 F. culmorum, 9 F. poae, 5 F. sambucinum and 3. F. graminearum) obtained from weeds were pathogenic to seedlings of the winter wheat cv. Mercia. The study identified 12 new previously undocumented hosts for Fusarium species.
Extensive literature has been published on weeds as altemative hosts for plant viruses (Duffus, 1971). Far less literature, however, is available on weeds as alternative hosts for fungal pathogens. Karunakaran et al. (1980) showed that the weed Clerodendron inforfunatum, commonly found growing in clove gardens, could act as an alternative host for the clove leaf-spot pathogen Colletofrichum gloeosporioides, and that isolates obtained from the weed were pathogenic to clove plants. Weeds have also been shown to provide an alternative host for the cucumber powdery mildew fungus Eysiphe cichoracearum (Stone, 1962). Hepperley ef al. (1980) demonstrated that a single weed species Abufilon fheophrasfi could provide an alternative host for three pathogens of soybean; Phomopsis sojae, Colletofrichum gloeosporioides and C. dematiurn var. truncata. The weed was also shown to harbour Fusarium, Alfemaria and Epicoccum species. Amaranthus spinosus, Leonotis nepafaefolia and Leonorus sibiricus are three other common weeds shown to cany isolates of Phomopsis spp. pathogenic to soybean (Cerkaukas ef al., 1983). Work on Fusarium species in weeds has mostly been concentrated on Fusarium oxysporum. For example, McDonald & Leach (1976) found that the sugar beet stalk-blight pathogen Fusarium oxysporum f. sp. befae could be isolated from plants of the weed Amaranfhus retroflexus, which showed disease symptoms characteristic of attack by a vascular pathogen. The same pathogen was also isolated from apparently healthy (symptomless) plants of the weeds Chenopodiurn album, Brassica nigra and Anethum graveolens. Isolates of F. oxysporum f. sp. betae obtained from all four weed species were shown t o be pathogenic to sugar beet. Amaranfhus refroflexus has also been shown to harbour the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, as have weed members of the genera Digifaria, Malva and
Oyzopsis (Katan, 1971). Isolates of F. oxysporum f. sp. lycopersici obtained from these weeds were shown to be pathogenic to tomato seedlings. Whilst screening 2 1 weed species belonging to 14 families as possible symptomless carriers of the chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri, Haware & Nene (1982)found that only Cardiospermum halicacabum, Convolvulw arvensis, Cyperus rofundis and Tribulus terresfris yielded the pathogen. However, isolates obtained from these weeds were shown to be non-pathogenic to chickpea. In a similar study camed out by Helbig & Carroll (1984), 16 out of 21 dicotyledonous weed species, commonly found in soybean fields, were found to be infected with the soybean pathogen F. oxysporum f. sp. glycines despite showing no disease symptom. Of 17 isolates of the pathogen tested, 16 were shown to be pathogenic to seedlings of the soybean cultivar Essex. Although Fusarium species pathogenic to winter wheat have been isolated from a range of crops and grasses (Gordon, 1959), little work has been documented on the incidence of these pathogens in common broad-leaved weeds. With rotational fallows being encouraged throughout European agriculture, this study set out to investigate the possibility of broad-leaved weeds as altemative hosts for Fusarium species pathogenic to winter wheat and to assess the role of such weeds in the survival of these pathogens.
MATERIALS A N D M E T H O D S
Field sfudies Three separate sites of fallow land, two following winter wheat crops and one following a crop of potatoes, were included in the study. Sites were located near Harper Adams
P. Jenkinson and D. W. Parry Agricultural College, Shropshire, U.K. A total of 1346 plants representing 15 weed species and 10 families were collected from the three sites: Beta vulgaris L. (volunteer crop plant), Capselb bursa-pasforis (L.) Medikus, Cirsium aruense (L.) Scop., Galium aparine L., Mafricaria L. spp., Ranunculus acris L., Ranunculus repens L., Rumex obfusifolius L., Senecio vulgaris L., Spergula aruensis L., Stellaria media L., Urfica dioica L., U r f ~ c a urens L., Veronica persica Poiret and Viola arvensis L. After collection, plants were sealed in polythene bags and transported to the laboratory, where they were prepared for the isolation of Fusarium species.
777 Table 1. Key used for the assessment of the severity of Fwarium seedling blight on winter wheat seedlings (cv. Mercia) infected with Fwarium isolates obtained from common broad-leaved weeds (after Malalasekera & Colhoun, 1968) Disease category 0 1
2 3
4 5
6
Symptom Healthy Small lesions on roots or stem-base Larger lesions on roots or stem-base Severe necrosis of stem-base, plants stunted and yellow Plants wilted and dying Post-emergence death of plant Pre-emergence death of plant
Disease Index =
Isolation of Fusarium
All plants were washed thoroughly for several minutes in running water before being visually inspected for possible symptoms of Fusarium infection. After being washed, a cylindrical segment (30 mm long) was cut transversely from the stem-base of each plant. Excised stem-bases were then surface-sterilized in a 4 % sodium hypochlorite solution (0.5 % available chlorine) for 3 min to reduce surface contaminants found on the outer stem surfaces. After rinsing in three changes of sterile distilled water, excised stem-bases were placed on to sterile filter paper and left for 30 min to dry at room temperature. When dry, stem-bases were placed in 90 mm Petri dishes containing Potato Dextrose Agar (PDA), two stems per dish. The PDA was amended with an antibiotic mixture consisting of streptomycin sulphate (10 pg ml-l), aureomycin (50 ~g ml-l) and chloramphenicol (50 pg ml-I). Stem-bases were incubated in darkness at 20 OC for 10 d, after which the incidence of each Fusarium species isolated was recorded. Those isolates which could not be identified as Fusarium species directly after incubation were sub-cultured on to 90 mm Petri dishes containing Sucrose Nutrient Agar (SNA) (Nirenberg, 1976), a medium low in nutrients and which induces the production of uniform spores. Since Fusarium isolates grow very sparsely on SNA and do not always produce pigmentation on it, isolates were also sub-cultured on to Potato Sucrose Agar (PSA).All sub-cultures were incubated at 20' under a 12 h lightldark regime for 10 d, after which the spore morphology (determined by microscopic exarnination of the SNA plate under x 25 magnification) and colony morphology and pigmentation (determined by visual inspection of PSA plate) was recorded for each isolate. From these characteristics, isolates were identified to species level according to Booth (1971).
Where a, b, c, d, e, f and g are the number of plants in each disease category.
colonies, a conidial spore suspension was prepared for each isolate by the addition of 5 ml of sterile distilled water. A sterile spatula was used to dislodge conidia from mycelium. Each spore suspension was then filtered through two layers of muslin to remove hyphal fragments and centrifuged at 3000 rpm for 3 min. Following centrifugation, the supernatant was decanted off and the remaining spores resuspended in sterile distilled water to obtain a clean conidial suspension. The concentration of each conidial suspension was then determined using a haemocytometer and adjusted to give 75 000 spores mlpl of water. Seeds of the winter wheat cultivar Mercia were surfacesterilized in a 4 % sodium hypochlorite solution (0.5 % available chlorine). After being rinsed in three changes of sterile distilled water, seeds were allowed to dry at room temperature for 30 min. For each isolate, 15 disinfected wheat seeds were then inoculated by soaking in 1 ml of spore suspension for 30 min. The inoculated seeds were then sown into 10 mm plastic pots containing steam-sterilized compost (John Innes No. 2) at a rate of five seeds per pot. Three replicate pots were set up for each isolate tested. The soaking of disinfected seed in 1 ml of sterile distilled water provided ten uninoculated control treatments, each with three replicates. All pots were arranged according to a random design on a greenhouse bench and incubated at 17k2O under a 12 h photoperiod for 4-5 wk. After 4 wk, when plants were at growth stage 4 (Zadoks ef al., 1974). wheat plants were harvested and washed in running water. Each plant was then visually inspected and the severity of Fusarium seedling blight assessed according to Malalasekera & Colhoun (1968) (Table I).From the assessment, a disease index score was then calculated for each isolate using Pathogenicity studies equation ( I ) (TabIe 1). Isolates giving disease index scores of Seventy-seven Fusarium isolates (33 F. avenaceum, 27 F. zero were considered non-pathogenic to wheat. In order to culmorum, 9 F. poae, 5 F. sambucinum and 3 F. grarninearum), verify that disease symptoms observed were a direct result of obtained from broad-leaved weeds, were tested for their artificial inoculation, Fusarium species were re-isolated from pathogenicity to seedlings of the winter wheat (Trificum infected wheat plants by placing surface-sterilized segments of aesfivum)cv. Mercia. Each isolate was sub-cultured on to SNA stem-base (30 mm long) on to 90 mrn Petri dishes containing and incubated at 20' under a 12 h darkllight regime until PDA. After incubating stem-bases at 20' for 10-14 d in the sporulating colonies were obtained. From these sporulating dark, isolates of Fusarium found growing were identified.
Fusarium in broad-leaved weeds
778
Table 2. The incidence of Fusarium species (expressed as percentage of stems infected) isolated from 1346 common broad-leaved weed plants collected from three separate sites of fallow No. of plants assessed Site A Compositae Ranunculaceae Polygonacae Rubiaceae Violaceae Site B Compositae Ranunculaceae Chenopodiaceae Cruciferae Caryophyllaceae Urticaceae Saophulariaceae Site C Compositae Cruciferae Rubiaceae Caryophyllaceae Urticaceae Violaceae
Cirsium arvense Senecio vulgaris Ranunculus acris R. repens Rumex obtwifolius Galium aparine Viola amensis
F. auenaceum
F. culmotum
F. poae
-
-
25
5.2
45 70 35 32 45 40
15.9 1.4 294 3.1 21.4 33.3
2.2 14.7 26-4 12.5 178 16.7
40
-
-
-
-
-
-
-
-
55.0 3.3 566 100
10.0 3.3 6.4
-
-
2.5
-
Matricaria spp. Senecio vulgaris Ranunculus repens Befa vulgaris Capsulla bursa-pastoris Spergula a m m e Stellaria media Urtica urens Veronica persica
120 60 82 43 38 55 80 124
Matricaria spp. Smecio vulgaris Capsella bursa-pastoris Galium aparine Stellaria media Urtica dioica Viola amensis
40 90 62 80 40 50 50
-
180 4.0
8-3 1-6 15-8
34.0 8.0
Other fusaria
-
14.3 11.1
-
-
-
5.2 18.1 17.7 55.8 15.6 53.5 61.1
A
-
1.5
-
Total
2.6
2.6 8.3 1.6 15.8
-
-
-
2-6
-
-
-
-
-
-
A
7.5 8.0 1.3
2.0
2-6
1.6
1.6
75
80.0 6.6 73.5 11.3 2.5 52.0 18.0
-
1.6
-
4.0
Table 3. Mean Fusarium disease index for seedlings of winter wheat (cv. Mercia) inoculated with isolates of Fwarium obtained from broad-leaved weeds
Isolate source
No. of isolates tested
Mean Fusanum disease indexa
F. avenaceum
Capsella bursa-pastoris Cirsiltm amense Smecio vulgaris Matricaria spp. Ranunculus am5 R. repens Rumex obtwifolius Galium aparine Stellaria media Urtica dioica Viola amensis
6
21.1 (2-4)
F. poae
Capsella bursa-pastoris Galium aparine Matricaria spp. Ranunculus acris Viola amensis
1
7.3 (1.8) 4.3 (1.2) 10.0 (1.9) 6.7 (2.1) 5.6 (2.9)
Controlb
3 2 I 2
0.0 (0.0)
Isolate source F. cuhorum
Beta vulgaris Capsella bursa-past01 Senecio vulgaris Matricaria spp. Ranunculus am's Ranunculus repens Rumex obtusifolius Galium aparine Urtica dioica Viola amensis
F. graminearum
Matricaria spp. Veronica persica Viola amensis Capsella bursa-pastoris Matricaria spp. Spergula aruensis Urtica dioica Viola amensis
F. sambucinum
No. of isolates tested 2
Mean Fusarium disease indexa 9 4 (1.8)
Based on three replicate pots (each containing five wheat seedlings) for each isolate tested. Fusarium index calculated according to Malalasekera & Colhoun (1968).
Ten control treatments (each consisting of three replicate pots containing five wheat seedlings) inoculated with sterile distilled water Numbers in parentheses are standard errors.
P. Jenlunson and D. W. Parry RESULTS No obvious symptom of Fusarium infection was seen on any of the weed species following visual inspection of sample plants. The incidence of Fusarium species in 15 weed species can be seen in Table 2. Isolations made from 1346 plants yielded 226 Fusarium isolates, of which 114 (50.4%) were F. avenaceum, 88 (38.9%) were F. cu/morum, 16 (7.1%) were F. poae, 5 (2.2%) were F. sambucinum and 3 (1.3%) were F. graminearum. Of the 5 species isolated, F. avenaceum and F. culmorum were the most widespread, having been isolated respectively from 11 and 10 of the 15 weed species studied. Fusarium poae and F. sambucinum were each isolated from five weed species, whilst F. graminearum was only isolated from mayweed (Matricaria spp.) and field pansy (Viola arvensis).Small nettle (Urfica urens) was the only weed species that failed to yield Fusarium. In the majority of cases, individual weed species were shown to harbour several Fusarium species, though not necessarily in the same plant. Mayweed (Matricaria spp.) and field pansy (Viola arvensis), for example, were infected by all five of the Fusarium species isolated, whilst shepherd's purse (Capsella bursa-pastoris) was infected by F. avenaceum, F. culmorum, F. poae and F. sambucinum. Only weed beet (Beta vulgaris), chickweed (Stellaria media), spurrey (Spergula arvensis) and common field speedwell (Veronica persica) were infected by a single species of Fusarium (Table 2). The incidence of weeds infected with Fusarium varied considerably between the three sites. Site B, which was a site of fallow following a crop of potatoes, showed a very low incidence of Fusarium-infected weeds, with only 15.8% of weed beet plants infected. The incidence of Fusarium-infected plants amongst other weed species was generally less than 5%. The only Fusarium species isolated from site B were F. culmorum and F. sambucinum. In contrast, a far higher incidence of Fusarium was observed in sites A and C, sites of fallow which followed wheat crops. At both these sites, the incidence of Fusarium-infected plants was > 50% in several weed species, with up to 80% of mayweed plants infected at site C. Fusarium species isolated from site A were F. avenaceum, F. culmorum and F. pone, of which F. avenaceum and F. culmorum were predominant, accounting for 48.3% and 3 9 8 % respectively of isolations made at this site. All five Fusarium species were isolated at site C, with F. avenaceum and F. culmorum again proving the most common, having accounted for 61.2% and 25.3% of isolations made respectively. Table 3 shows the severity of Fusarium seedling blight in wheat following artificial inoculation with Fusarium isolates obtained from weeds. Of 77 isolates tested (33 F. avenaceum, 27 F. culmorum, 9 F. poae, 5 F. sambucinum and 3 F. grarninearum), 75 were pathogenic to seedlings of winter wheat. Fusarium avenaceum, F. culmorum and F. graminearum were the most pathogenic of the five species, generally resulting in mean Fusarium disease indices of between 20 and 30 depending on the origin of the isolate. Isolates of F. poae and F. sambucinurn isolated from seeds were weakly pathogenic to wheat seedlings, resulting in Fusariurn disease indices of less than 10. No symptom of Fusarium infection was observed on any of the uninoculated control plants. Reisolation of
Table 4. New hosts identified for F. avenaceum, F. culmorum. F. graminearum, F. poae and F. sambucinum
Host
F. auenaceum
F. culmorum
F. graminearum F. poae
F. sambwinum
Capsella bursa-pastoris Cirsium aruense Matricaria spp Ranunculus acris R. repens Rumex obtusifolius Stellaria media Urfica dioica Viola awmsis Capsella bursa-pastoris Galium aparine Matricaria spp. Ranunnrlus am's R. repms Rumex obtusifolius Urfica dioica Viola arvensis Matricaria spp. Viola awmsis Capsella bursa-pasforis Galium aparine Marticaria spp. Ranunculus acris Viola awensis Capsella bursa-pastoris Matricaria spp. Spergula arvensis Veronica persica
Fusarium species from infected plants showed that all infections were a result of artificial inoculation. Table 4 summarizes those weed species which were identified as new, previously undocumented hosts for each of the five Fusarium species isolated. In all, 9 new hosts were identified for F. avenaceum, 8 for F. culmorum, 5 for F. poae, 4 for F. sambucinum and 2 for F. graminearum.
DISCUSSION Common weeds have been demonstrated to serve as alternative hosts for several diseases of cultivated plants (Dinoor, 1974). In this study, several common broad-leaved weeds were clearly shown to provide alternative hosts for F. avenaceum, F. culmorum, F. graminearum, F. poae and F. sambucinum (Table 2), and isolates harboured by these weeds were shown to be pathogenic to winter wheat (Table 3). The study also identified several new undocumented hosts for each of five Fusarium species isolated (Table 4). Assuming that the sodium hypochlorite eradicated all surface contaminants, any Fusarium isolated from the weeds must have originated from infected stem tissues. Obviously the efficacy of sodium hypochlorite as a surface sterilant will not be 100%. and further microscopic work would be necessary to determine exactly where in the stem of weeds the Fusarium was located. If, however, such studies revealed the successful infection of plant tissue, the lack of symptoms observed on weeds would suggest that the infection of these hosts by Fusarium species may be endophytic. The endophytic
Fusarium in broad-leaved weeds infection of wheat by F. culrnorum, F. graminearurn and Microdochium nivale (formerly Fusariurn nivale) has been suggested by Sieber ef al. (1988). Symptomless infection has also been observed in several weed species infected with F. oxysporum f. sp. betae (MacDonald & Leach, 1976), F. oxysporum f. sp. lycopersici (Katan, 1971), F. oxysporum f. sp. ciceri (Haware & Nene, 1982) and F. oxysporum f. sp. glycines (Helbig & Carroll, 1984). The frequent isolation of Fusarium from common weeds raises the question of their role in the survival of the fungus. Fusarium avenaceum, F. culrnorum, F. graminearurn and F. sambucinum can all survive saprotrophically on crop debris or in soil as resting spores (chlamydospores). The relative importance of weeds in the survival of these pathogens can therefore only be speculated. Fusarium poae, however, has not been observed to produce chlarnydospores. The fungus is rarely isolated from either crop debris or wheat culms, and yet is the species most commonly isolated from the ears of winter wheat in England and Wales (Polley et al., 1991). It would appear, then, that weeds could provide an important alternative host for the survival of F. poae. As well as providing a means of survival, common weeds could provide an important source of Fusariurn inoculum for the infection of wheat crops. The importance of weeds as reservoirs of potential fungal pathogens was recognized by Garrett (1960) when he suggested that the roots of weeds infected by a particular soil-borne pathogen could act as direct inoculum for the roots of any susceptible following crop. Although tests showed that Fusarium species from weeds were pathogenic to wheat seedlings, the pathogenicity of these isolates was not compared to that of isolates obtained from wheat. Consequently, the relative importance of weeds as sources of inoculum for the development of Fusarium diseases in wheat cannot be determined from this study. Clearly the study has shown that common broad-leaved weeds can harbour Fusarium species pathogenic to wheat and has identified 12 new hosts for cereal fusaria. However, further work is needed if the epidemiological significance of Fusarium-infected weeds is to be understood.
The authors acknowledge Dr P. E. Russell of Schering Agrochemicals and the Science and Engineering Research Council for the financial support of this study. We thank Dr
780 Tom Preece and Dr Tim Pettitt for their invaluable comments during the preparation of this manuscript.
REFERENCES Booth, C. (1971). The Genw Fusarium. Commonwealth Agricullural Bureaux: London. Cerkauskas, R. F., Dhingra, 0.D., Sinclair, J. B. & Asmus, G. (1983). Amaranthw spinosw, Leonotis nepetaefolia and Leononcs sibiricw: new hosts of Phomopsis spp. in Brazil. Plant Disease 67,821-824. Dinoor, A. (1974). Role of wild and cultivated plants in the epidemiology of plant disease in Israel. Annual Review of Phytopathology 12,413-436. Duffus, J. E. (1971). Role of weeds in the incidence of virus diseases. Annual Review of Phytopathology 9, 319-340. Garrett, S. D. (1960). Inoculurn potential. In Plant Pathology. A n Advanced Treatise, vol. 3 (ed. J. G. Horsfall & A. E. Disrnond), pp. 23-56. Academic Press: New York. Gordon. W. L. (1959). The occurrence of Fwarium species in Canada. VI. Taxonomy and geographic distribution of Fusarium species on plants, insects and fungi. Canadian ]ournal of Botany 37,257-290. Haware, M. P. & Nene, Y. L. (1982). Symptomless carriers of the chickpea wilt seedling Fwarium. Plant Disease 66,250-251. Helbig, J. B. & Carroll, R. 8.(1984). Dicotyledonous weeds as a source of Fwarium oxysporum pathogenic on soybean. Plant Disease 68,694496. Hepperley, P. R., Kirkpatrick, B. L. & Sinclair, J. B. (1980).Abutilon theophrasti: wild host for three fungal parasites of soybean. Phytopathology 70, 307-310. Karunakaran, P. Chandrasekharan Nair, M. & Gokulapalan, C. (1980).Survival of the clove pathogen Colletotrichumgloeosporioideson the weed Clerodendron in India. Plant Disease 64,415-416. Katan, J. (1971). Symptomless caniers of the tomato Fusarium wilt pathogen. Phyt~~athology 61, 1213-1217. MacDonald, J. D. & Leach, L. D. (1976). Evidence for an expanded host range of Fwarium oxysporum f. sp. betae. Phytopathology 66,822-827. Malalasekera, R. A. P. & Colhoun J.(1968). Fusariurn diseases of cereals. V. A technique for the examination of wheat seed infected with Fwarium culmorum. Transactions of the British Mycological Sociely 51,711-720. Nirenberg, H. (1976). Untersuchungen uber die rnorphologische und biologische Differenzierung in der Fwarium - Section Liseola. Mitteilungen aus der biologischen Bundersanstalt fur Land- und Forstwirtschaft 169,1-117. Polley, R. W., Turner, J. A., Cockerell, V.,Robb, J., Scudamore, K. A.. Sanders, M. F. & Magan, N. (1991). Survey of Ftrsarium species infecting winter wheat in England, Wales and Scotland, 1989-1990. HGCA Project Report No. 39. Sieber. T., Risen, T. K., Miiller, E. & Fried, P. M. (1988). Endophytic fungi in four winter wheat cultivars (Triticum aestivum L.) differing in resistance against Stagonospora nodorum (Berk.) Cast. & Germ. = Septoria nodorum (Berk.) Berk. Journal of Phytop~tholo~y 122,289-306. Stone, 0.M. (1962). Alternate hosts of cucumber powdery mildew. Annals of Applied Biology 50,203-210. Zadoks, J. C., Chang, T. T. & Konzak, C. F. (1974). A decimal code for the growth stages of cereals. Weed Research 14,415-421.
Mycol. Res. 98 (7): 7 7 6 7 8 0 (1994) Printed in Great Brifain
Isolation of Fusarium species from common broad-leaved weeds and their pathogenicity to winter wheat
P. J E N K I N S O N A N D D. W. PARRY Crop and Environment Research Centre, Harper Adams Agricultural College, Newport, Shropshire, TFlO 8NB. U.K.
Isolations were made from the stem-bases of 1346 plants representing 15 weed species collected from three sites of fallow land; one following a crop of potatoes and two following wheat crops. Two hundred and twenty-six Fwarium isolates were obtained from 14 of the 15 weed species of which 114 (50.4%) were F. avenaceum, 88 (38.9%) were F. culmorum, 16 (7.1%)were F. poae, 6 (2.5%) were F. sambucinum and 3 (1.3%) were F. graminearum. No obvious symptom of Fusarium infection was observed on any of the plants sampled. Seventy-five of 77 Fusarium isolates (33 F. avenaceum, 27 F. culmorum, 9 F. poae, 5 F. sambucinum and 3. F. graminearum) obtained from weeds were pathogenic to seedlings of the winter wheat cv. Mercia. The study identified 12 new previously undocumented hosts for Fusarium species.
Extensive literature has been published on weeds as altemative hosts for plant viruses (Duffus, 1971). Far less literature, however, is available on weeds as alternative hosts for fungal pathogens. Karunakaran et al. (1980) showed that the weed Clerodendron inforfunatum, commonly found growing in clove gardens, could act as an alternative host for the clove leaf-spot pathogen Colletofrichum gloeosporioides, and that isolates obtained from the weed were pathogenic to clove plants. Weeds have also been shown to provide an alternative host for the cucumber powdery mildew fungus Eysiphe cichoracearum (Stone, 1962). Hepperley ef al. (1980) demonstrated that a single weed species Abufilon fheophrasfi could provide an alternative host for three pathogens of soybean; Phomopsis sojae, Colletofrichum gloeosporioides and C. dematiurn var. truncata. The weed was also shown to harbour Fusarium, Alfemaria and Epicoccum species. Amaranthus spinosus, Leonotis nepafaefolia and Leonorus sibiricus are three other common weeds shown to cany isolates of Phomopsis spp. pathogenic to soybean (Cerkaukas ef al., 1983). Work on Fusarium species in weeds has mostly been concentrated on Fusarium oxysporum. For example, McDonald & Leach (1976) found that the sugar beet stalk-blight pathogen Fusarium oxysporum f. sp. befae could be isolated from plants of the weed Amaranfhus retroflexus, which showed disease symptoms characteristic of attack by a vascular pathogen. The same pathogen was also isolated from apparently healthy (symptomless) plants of the weeds Chenopodiurn album, Brassica nigra and Anethum graveolens. Isolates of F. oxysporum f. sp. betae obtained from all four weed species were shown t o be pathogenic to sugar beet. Amaranfhus refroflexus has also been shown to harbour the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, as have weed members of the genera Digifaria, Malva and
Oyzopsis (Katan, 1971). Isolates of F. oxysporum f. sp. lycopersici obtained from these weeds were shown to be pathogenic to tomato seedlings. Whilst screening 2 1 weed species belonging to 14 families as possible symptomless carriers of the chickpea wilt pathogen Fusarium oxysporum f. sp. ciceri, Haware & Nene (1982)found that only Cardiospermum halicacabum, Convolvulw arvensis, Cyperus rofundis and Tribulus terresfris yielded the pathogen. However, isolates obtained from these weeds were shown to be non-pathogenic to chickpea. In a similar study camed out by Helbig & Carroll (1984), 16 out of 21 dicotyledonous weed species, commonly found in soybean fields, were found to be infected with the soybean pathogen F. oxysporum f. sp. glycines despite showing no disease symptom. Of 17 isolates of the pathogen tested, 16 were shown to be pathogenic to seedlings of the soybean cultivar Essex. Although Fusarium species pathogenic to winter wheat have been isolated from a range of crops and grasses (Gordon, 1959), little work has been documented on the incidence of these pathogens in common broad-leaved weeds. With rotational fallows being encouraged throughout European agriculture, this study set out to investigate the possibility of broad-leaved weeds as altemative hosts for Fusarium species pathogenic to winter wheat and to assess the role of such weeds in the survival of these pathogens.
MATERIALS A N D M E T H O D S
Field sfudies Three separate sites of fallow land, two following winter wheat crops and one following a crop of potatoes, were included in the study. Sites were located near Harper Adams
P. Jenkinson and D. W. Parry Agricultural College, Shropshire, U.K. A total of 1346 plants representing 15 weed species and 10 families were collected from the three sites: Beta vulgaris L. (volunteer crop plant), Capselb bursa-pasforis (L.) Medikus, Cirsium aruense (L.) Scop., Galium aparine L., Mafricaria L. spp., Ranunculus acris L., Ranunculus repens L., Rumex obfusifolius L., Senecio vulgaris L., Spergula aruensis L., Stellaria media L., Urfica dioica L., U r f ~ c a urens L., Veronica persica Poiret and Viola arvensis L. After collection, plants were sealed in polythene bags and transported to the laboratory, where they were prepared for the isolation of Fusarium species.
777 Table 1. Key used for the assessment of the severity of Fwarium seedling blight on winter wheat seedlings (cv. Mercia) infected with Fwarium isolates obtained from common broad-leaved weeds (after Malalasekera & Colhoun, 1968) Disease category 0 1
2 3
4 5
6
Symptom Healthy Small lesions on roots or stem-base Larger lesions on roots or stem-base Severe necrosis of stem-base, plants stunted and yellow Plants wilted and dying Post-emergence death of plant Pre-emergence death of plant
Disease Index =
Isolation of Fusarium
All plants were washed thoroughly for several minutes in running water before being visually inspected for possible symptoms of Fusarium infection. After being washed, a cylindrical segment (30 mm long) was cut transversely from the stem-base of each plant. Excised stem-bases were then surface-sterilized in a 4 % sodium hypochlorite solution (0.5 % available chlorine) for 3 min to reduce surface contaminants found on the outer stem surfaces. After rinsing in three changes of sterile distilled water, excised stem-bases were placed on to sterile filter paper and left for 30 min to dry at room temperature. When dry, stem-bases were placed in 90 mm Petri dishes containing Potato Dextrose Agar (PDA), two stems per dish. The PDA was amended with an antibiotic mixture consisting of streptomycin sulphate (10 pg ml-l), aureomycin (50 ~g ml-l) and chloramphenicol (50 pg ml-I). Stem-bases were incubated in darkness at 20 OC for 10 d, after which the incidence of each Fusarium species isolated was recorded. Those isolates which could not be identified as Fusarium species directly after incubation were sub-cultured on to 90 mm Petri dishes containing Sucrose Nutrient Agar (SNA) (Nirenberg, 1976), a medium low in nutrients and which induces the production of uniform spores. Since Fusarium isolates grow very sparsely on SNA and do not always produce pigmentation on it, isolates were also sub-cultured on to Potato Sucrose Agar (PSA).All sub-cultures were incubated at 20' under a 12 h lightldark regime for 10 d, after which the spore morphology (determined by microscopic exarnination of the SNA plate under x 25 magnification) and colony morphology and pigmentation (determined by visual inspection of PSA plate) was recorded for each isolate. From these characteristics, isolates were identified to species level according to Booth (1971).
Where a, b, c, d, e, f and g are the number of plants in each disease category.
colonies, a conidial spore suspension was prepared for each isolate by the addition of 5 ml of sterile distilled water. A sterile spatula was used to dislodge conidia from mycelium. Each spore suspension was then filtered through two layers of muslin to remove hyphal fragments and centrifuged at 3000 rpm for 3 min. Following centrifugation, the supernatant was decanted off and the remaining spores resuspended in sterile distilled water to obtain a clean conidial suspension. The concentration of each conidial suspension was then determined using a haemocytometer and adjusted to give 75 000 spores mlpl of water. Seeds of the winter wheat cultivar Mercia were surfacesterilized in a 4 % sodium hypochlorite solution (0.5 % available chlorine). After being rinsed in three changes of sterile distilled water, seeds were allowed to dry at room temperature for 30 min. For each isolate, 15 disinfected wheat seeds were then inoculated by soaking in 1 ml of spore suspension for 30 min. The inoculated seeds were then sown into 10 mm plastic pots containing steam-sterilized compost (John Innes No. 2) at a rate of five seeds per pot. Three replicate pots were set up for each isolate tested. The soaking of disinfected seed in 1 ml of sterile distilled water provided ten uninoculated control treatments, each with three replicates. All pots were arranged according to a random design on a greenhouse bench and incubated at 17k2O under a 12 h photoperiod for 4-5 wk. After 4 wk, when plants were at growth stage 4 (Zadoks ef al., 1974). wheat plants were harvested and washed in running water. Each plant was then visually inspected and the severity of Fusarium seedling blight assessed according to Malalasekera & Colhoun (1968) (Table I).From the assessment, a disease index score was then calculated for each isolate using Pathogenicity studies equation ( I ) (TabIe 1). Isolates giving disease index scores of Seventy-seven Fusarium isolates (33 F. avenaceum, 27 F. zero were considered non-pathogenic to wheat. In order to culmorum, 9 F. poae, 5 F. sambucinum and 3 F. grarninearum), verify that disease symptoms observed were a direct result of obtained from broad-leaved weeds, were tested for their artificial inoculation, Fusarium species were re-isolated from pathogenicity to seedlings of the winter wheat (Trificum infected wheat plants by placing surface-sterilized segments of aesfivum)cv. Mercia. Each isolate was sub-cultured on to SNA stem-base (30 mm long) on to 90 mrn Petri dishes containing and incubated at 20' under a 12 h darkllight regime until PDA. After incubating stem-bases at 20' for 10-14 d in the sporulating colonies were obtained. From these sporulating dark, isolates of Fusarium found growing were identified.
Fusarium in broad-leaved weeds
778
Table 2. The incidence of Fusarium species (expressed as percentage of stems infected) isolated from 1346 common broad-leaved weed plants collected from three separate sites of fallow No. of plants assessed Site A Compositae Ranunculaceae Polygonacae Rubiaceae Violaceae Site B Compositae Ranunculaceae Chenopodiaceae Cruciferae Caryophyllaceae Urticaceae Saophulariaceae Site C Compositae Cruciferae Rubiaceae Caryophyllaceae Urticaceae Violaceae
Cirsium arvense Senecio vulgaris Ranunculus acris R. repens Rumex obtwifolius Galium aparine Viola amensis
F. auenaceum
F. culmotum
F. poae
-
-
25
5.2
45 70 35 32 45 40
15.9 1.4 294 3.1 21.4 33.3
2.2 14.7 26-4 12.5 178 16.7
40
-
-
-
-
-
-
-
-
55.0 3.3 566 100
10.0 3.3 6.4
-
-
2.5
-
Matricaria spp. Senecio vulgaris Ranunculus repens Befa vulgaris Capsulla bursa-pastoris Spergula a m m e Stellaria media Urtica urens Veronica persica
120 60 82 43 38 55 80 124
Matricaria spp. Smecio vulgaris Capsella bursa-pastoris Galium aparine Stellaria media Urtica dioica Viola amensis
40 90 62 80 40 50 50
-
180 4.0
8-3 1-6 15-8
34.0 8.0
Other fusaria
-
14.3 11.1
-
-
-
5.2 18.1 17.7 55.8 15.6 53.5 61.1
A
-
1.5
-
Total
2.6
2.6 8.3 1.6 15.8
-
-
-
2-6
-
-
-
-
-
-
A
7.5 8.0 1.3
2.0
2-6
1.6
1.6
75
80.0 6.6 73.5 11.3 2.5 52.0 18.0
-
1.6
-
4.0
Table 3. Mean Fusarium disease index for seedlings of winter wheat (cv. Mercia) inoculated with isolates of Fwarium obtained from broad-leaved weeds
Isolate source
No. of isolates tested
Mean Fusanum disease indexa
F. avenaceum
Capsella bursa-pastoris Cirsiltm amense Smecio vulgaris Matricaria spp. Ranunculus am5 R. repens Rumex obtwifolius Galium aparine Stellaria media Urtica dioica Viola amensis
6
21.1 (2-4)
F. poae
Capsella bursa-pastoris Galium aparine Matricaria spp. Ranunculus acris Viola amensis
1
7.3 (1.8) 4.3 (1.2) 10.0 (1.9) 6.7 (2.1) 5.6 (2.9)
Controlb
3 2 I 2
0.0 (0.0)
Isolate source F. cuhorum
Beta vulgaris Capsella bursa-past01 Senecio vulgaris Matricaria spp. Ranunculus am's Ranunculus repens Rumex obtusifolius Galium aparine Urtica dioica Viola amensis
F. graminearum
Matricaria spp. Veronica persica Viola amensis Capsella bursa-pastoris Matricaria spp. Spergula aruensis Urtica dioica Viola amensis
F. sambucinum
No. of isolates tested 2
Mean Fusarium disease indexa 9 4 (1.8)
Based on three replicate pots (each containing five wheat seedlings) for each isolate tested. Fusarium index calculated according to Malalasekera & Colhoun (1968).
Ten control treatments (each consisting of three replicate pots containing five wheat seedlings) inoculated with sterile distilled water Numbers in parentheses are standard errors.
P. Jenlunson and D. W. Parry RESULTS No obvious symptom of Fusarium infection was seen on any of the weed species following visual inspection of sample plants. The incidence of Fusarium species in 15 weed species can be seen in Table 2. Isolations made from 1346 plants yielded 226 Fusarium isolates, of which 114 (50.4%) were F. avenaceum, 88 (38.9%) were F. cu/morum, 16 (7.1%) were F. poae, 5 (2.2%) were F. sambucinum and 3 (1.3%) were F. graminearum. Of the 5 species isolated, F. avenaceum and F. culmorum were the most widespread, having been isolated respectively from 11 and 10 of the 15 weed species studied. Fusarium poae and F. sambucinum were each isolated from five weed species, whilst F. graminearum was only isolated from mayweed (Matricaria spp.) and field pansy (Viola arvensis).Small nettle (Urfica urens) was the only weed species that failed to yield Fusarium. In the majority of cases, individual weed species were shown to harbour several Fusarium species, though not necessarily in the same plant. Mayweed (Matricaria spp.) and field pansy (Viola arvensis), for example, were infected by all five of the Fusarium species isolated, whilst shepherd's purse (Capsella bursa-pastoris) was infected by F. avenaceum, F. culmorum, F. poae and F. sambucinum. Only weed beet (Beta vulgaris), chickweed (Stellaria media), spurrey (Spergula arvensis) and common field speedwell (Veronica persica) were infected by a single species of Fusarium (Table 2). The incidence of weeds infected with Fusarium varied considerably between the three sites. Site B, which was a site of fallow following a crop of potatoes, showed a very low incidence of Fusarium-infected weeds, with only 15.8% of weed beet plants infected. The incidence of Fusarium-infected plants amongst other weed species was generally less than 5%. The only Fusarium species isolated from site B were F. culmorum and F. sambucinum. In contrast, a far higher incidence of Fusarium was observed in sites A and C, sites of fallow which followed wheat crops. At both these sites, the incidence of Fusarium-infected plants was > 50% in several weed species, with up to 80% of mayweed plants infected at site C. Fusarium species isolated from site A were F. avenaceum, F. culmorum and F. pone, of which F. avenaceum and F. culmorum were predominant, accounting for 48.3% and 3 9 8 % respectively of isolations made at this site. All five Fusarium species were isolated at site C, with F. avenaceum and F. culmorum again proving the most common, having accounted for 61.2% and 25.3% of isolations made respectively. Table 3 shows the severity of Fusarium seedling blight in wheat following artificial inoculation with Fusarium isolates obtained from weeds. Of 77 isolates tested (33 F. avenaceum, 27 F. culmorum, 9 F. poae, 5 F. sambucinum and 3 F. grarninearum), 75 were pathogenic to seedlings of winter wheat. Fusarium avenaceum, F. culmorum and F. graminearum were the most pathogenic of the five species, generally resulting in mean Fusarium disease indices of between 20 and 30 depending on the origin of the isolate. Isolates of F. poae and F. sambucinurn isolated from seeds were weakly pathogenic to wheat seedlings, resulting in Fusariurn disease indices of less than 10. No symptom of Fusarium infection was observed on any of the uninoculated control plants. Reisolation of
Table 4. New hosts identified for F. avenaceum, F. culmorum. F. graminearum, F. poae and F. sambucinum
Host
F. auenaceum
F. culmorum
F. graminearum F. poae
F. sambwinum
Capsella bursa-pastoris Cirsium aruense Matricaria spp Ranunculus acris R. repens Rumex obtusifolius Stellaria media Urfica dioica Viola awmsis Capsella bursa-pastoris Galium aparine Matricaria spp. Ranunnrlus am's R. repms Rumex obtusifolius Urfica dioica Viola arvensis Matricaria spp. Viola awmsis Capsella bursa-pasforis Galium aparine Marticaria spp. Ranunculus acris Viola awensis Capsella bursa-pastoris Matricaria spp. Spergula arvensis Veronica persica
Fusarium species from infected plants showed that all infections were a result of artificial inoculation. Table 4 summarizes those weed species which were identified as new, previously undocumented hosts for each of the five Fusarium species isolated. In all, 9 new hosts were identified for F. avenaceum, 8 for F. culmorum, 5 for F. poae, 4 for F. sambucinum and 2 for F. graminearum.
DISCUSSION Common weeds have been demonstrated to serve as alternative hosts for several diseases of cultivated plants (Dinoor, 1974). In this study, several common broad-leaved weeds were clearly shown to provide alternative hosts for F. avenaceum, F. culmorum, F. graminearum, F. poae and F. sambucinum (Table 2), and isolates harboured by these weeds were shown to be pathogenic to winter wheat (Table 3). The study also identified several new undocumented hosts for each of five Fusarium species isolated (Table 4). Assuming that the sodium hypochlorite eradicated all surface contaminants, any Fusarium isolated from the weeds must have originated from infected stem tissues. Obviously the efficacy of sodium hypochlorite as a surface sterilant will not be 100%. and further microscopic work would be necessary to determine exactly where in the stem of weeds the Fusarium was located. If, however, such studies revealed the successful infection of plant tissue, the lack of symptoms observed on weeds would suggest that the infection of these hosts by Fusarium species may be endophytic. The endophytic
Fusarium in broad-leaved weeds infection of wheat by F. culrnorum, F. graminearurn and Microdochium nivale (formerly Fusariurn nivale) has been suggested by Sieber ef al. (1988). Symptomless infection has also been observed in several weed species infected with F. oxysporum f. sp. betae (MacDonald & Leach, 1976), F. oxysporum f. sp. lycopersici (Katan, 1971), F. oxysporum f. sp. ciceri (Haware & Nene, 1982) and F. oxysporum f. sp. glycines (Helbig & Carroll, 1984). The frequent isolation of Fusarium from common weeds raises the question of their role in the survival of the fungus. Fusarium avenaceum, F. culrnorum, F. graminearurn and F. sambucinum can all survive saprotrophically on crop debris or in soil as resting spores (chlamydospores). The relative importance of weeds in the survival of these pathogens can therefore only be speculated. Fusarium poae, however, has not been observed to produce chlarnydospores. The fungus is rarely isolated from either crop debris or wheat culms, and yet is the species most commonly isolated from the ears of winter wheat in England and Wales (Polley et al., 1991). It would appear, then, that weeds could provide an important alternative host for the survival of F. poae. As well as providing a means of survival, common weeds could provide an important source of Fusariurn inoculum for the infection of wheat crops. The importance of weeds as reservoirs of potential fungal pathogens was recognized by Garrett (1960) when he suggested that the roots of weeds infected by a particular soil-borne pathogen could act as direct inoculum for the roots of any susceptible following crop. Although tests showed that Fusarium species from weeds were pathogenic to wheat seedlings, the pathogenicity of these isolates was not compared to that of isolates obtained from wheat. Consequently, the relative importance of weeds as sources of inoculum for the development of Fusarium diseases in wheat cannot be determined from this study. Clearly the study has shown that common broad-leaved weeds can harbour Fusarium species pathogenic to wheat and has identified 12 new hosts for cereal fusaria. However, further work is needed if the epidemiological significance of Fusarium-infected weeds is to be understood.
The authors acknowledge Dr P. E. Russell of Schering Agrochemicals and the Science and Engineering Research Council for the financial support of this study. We thank Dr
780 Tom Preece and Dr Tim Pettitt for their invaluable comments during the preparation of this manuscript.
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