Effect of substrate and plant maturity on the incidence of infection of potato roots by pathogenic and non-pathogenic fungi

Mycol. Res. 9 7 (6):733-745 (1993)

733

Printed in Great Britain

Effect of substrate and plant maturity on the incidence of infection of potato roots by pathogenic and non-pathogenic fungi

E. P . D A S H W O O D , R . A . FOX A N D J. M. D U N C A N Scottish Crop Research Institute, Invergowrie, Dundee 0 0 2 SDA, LI.K

Colonization of roots of field-grown potato plants by pathogenic and non-pathogenic fungi was examined by plating surfacesterilized roots onto low nutrient medium. Plants which originated from micropropagated nodal cuttings (MP plants) or from seedgrade potato tubers (ST plants) were subjected to nine pre-plant treatments designed to test the effect of different potting substrates and different levels of natural tuber inoculum on the root mycoflora. The experiment was repeated in a second year but at a different site; in each year roots were collected once in July, August and September. Cylindrocarpon destructans, Colletotrichum coccodes, Trichoderma viride, Microdochium bolleyi and Fusarium fabacinum showed the highest mean frequencies of colonization; Verticillium kicorpus, Rhiwctonia solani, Polyscytalum pwtulans and Gliocladium roseum were mainly confined to roots of ST plants or MP plants with added tuber inoculum, and Trichoderma viride, Chysosporium pannorum, Penicillium spp., Aureobasidium pullulans and Phoma kveillei were dominant in roots of MP plants raised in peat-based potting substrates. There was evidence of suppression of the potato pathogens R. solani, P. pustulans and C. coccodes, and of several nonpathogens in roots of the same plants. Most of the fungi recovered from potato roots were also typical components of the soil microflora and occurred in both field sites. Exceptions were Fusarium oxysporum and V. dahliae, which were found in one year only. Possible associations and dissociations between different combinations of fungi occurring together on the same root system were evaluated using a correlation matrix, and spatial relationships of different groups demonstrated by principal coordinate analysis. The influence of the indigenous mycoflora on root colonization by particular potato tuber pathogens is discussed.

Infection of potato roots by fungal pathogens may result in poor plant emergence and growth, early senescence and reduced tuber yield. Colonized roots may act as a source of inoculum for tuber diseases; for example, Polyscytalum pwfulans, which causes skin spot in tubers, may sporulate from lesions on the root system during growth (Hide & Adams, 1980). Roots bearing rnicrosclerotia of the same fungus may also act as sunrival propagules for up to 7 yr (Hirst et al., 1970).The best-known pathogens of potato roots in temperate regions are Verficillium albo-atrum and V . dahliae, which invade the vascular systems and induce wilting and senescence, but some tuber pathogens, e.g. Rhizoctonia solani (black scurf), Collefofrichurn coccodes (black dot) and P. pustulans may cause local rotting or root-pruning (Hirst & Salt, 1959; Jellis & Taylor, 1974; Spencer & Fox, 1979). Others, e.g. Helminthosporium solani (silver scurf), Phoma foveata (gangrene) and Fwarium spp. (dry rot) have been isolated from potato roots but appear to do them little damage (Fox & Dashwood, 1982; Tivoli et al., 1988; Dashwood et al., 1992) Of primary concern to the potato seed producer in Scotland are those pathogens which may survive in soil over the minimum statutory gap of five years between potato crops. Long-term survival in the absence of the host has been demonstrated for P. pustulans, P. foveata, F. solani var. coeruleum (Carnegie & Cameron, 1990), C. coccodes (BIakeman & Hornby, 1966) and R. solani by direct soil sampling or bait tests (Herzog & Wartenberg 1958).

In Scotland, seed-grade tubers free from tuber-borne blemish and rot fungi are produced by micropropagation techniques which can produce seed-grade tubers from minitubers after only 3 yr of multiplication in the field. During this period the plants are at risk of infection by soil-bome inocula. Early contamination of progeny tubers, though slight, can progress to unacceptable levels of pathogen infection in the seed after several successive cultivations and storage periods. Camegie & Cameron (1990) described the relationship between the soil population levels of P. pustulans, P. foveata and F. s. var. coeruleum and tuber infection in axenically raised microplants. The relative importance of seed-borne over soilborne inoculum for these and other tuber pathogens has been emphasized by many authors, and discussed in detail by Adams & Hide (1980). However, comparatively little is known about root-borne inoculum of healthy potato plants, that is, the proportion of pathogens present in the root mycoflora and their distribution in the roots. Roots penetrate a greater soil mass and are exposed to soil inoculum for a longer period than are the progeny tubers. As well as acting as an infection base for tubers, there is evidence that infected roots or root fragments may contaminate tubers during harvesting- or in store (Hide & Adams, 1980). Colonization of roots by potentially harmful fungi is probably restricted by prior colonization or competition by the indigenous soil microflora, which is in intimate contact with the roots. The pathogens have to encounter this microflora

Mycoflora of roots of potato plants

734

before they can infect the root. It is therefore important to identify the more important component fungi of the community and their effects on the pathogens, whether antagonistic or associative. A knowledge of the conditions conducive for the multiplication of natural antagonists could be of practical value in the management of potato seed production. In an earlier paper Dashwood ef al. (1992) showed the effect of various levels of natural tuber inoculurn and different root substrates on the incidence of potato blemish pathogens on apparently healthy roots of micropropagated (MP) and seed tuber (ST) plants. The roots were surface-sterilized and examined on three dates in the growing season in two field experiments. This paper describes the distribution and incidence of the major groups of non-pathogenic fungi recovered from the same roots. The purpose of the study was to assess the effects of plant maturity and root substrate on the relative frequency of occurrence of pathogens and nonpathogens, and to assess the significance of any promising negative associations.

MATERIALS A N D M E T H O D S Micropropagated axenic plants and seed tubers cv. Maris Piper were planted in field experiments at Invergowrie in 1985 and 1986. Before planting they were subjected to nine treatments designed to differentiate between tuber- and soilborne sources of root infection and the effect of peat-based substrates (Table 1). Production and treatment of micropropagated plants

Microplants were produced from nodal stem cuttings and transplanted to three potting substrates, peat, UC compost and perlite as described earlier (Dashwood ef al., 1991). All plants were kept in a glasshouse at ca 15 OC for 4-5 wk, then hardened off outdoors for 1 wk before transfer to the field. Selection and treatment of seed tubers

Tubers of uniform size were selected from two certified seed stocks of cv. Maris Piper. One stock was free of symptoms ('clean stock') and the other was moderately infected by common blemish diseases ('blemished stock'). The fungi recorded on the latter in 1986 are listed in Table 2. Half the sample from each stock (treatments 7 and 9) were dipped in 1.0% NaOCl solution for 10 min to reduce surface conTable I. List of treatments

Treatment Potting number medium

Tuber inoculum

Plants grown from axenic plantlets I Perlite 2 Perlite

+

3 4 5

Peat Peat UC compost -

+

Treatment Seed tuber health and number treatment Plants grown from seed tubers 6 'Blemished' tubers 7 'Blemished' tubers with Chloros 8

'Clean ' tubers

9

'Clean' tubers treated with Chloros

tamination. All tubers were chitted in a cool glasshouse for 5 wk prior to planting. Planting and crop management

The experimental layout and management of the crop are described elsewhere (Dashwood ef al., 1991).Micropropagated plants (MP) and seed tubers (ST) were planted at Invergowrie on free-draining sandy loam soil. Different sites at SCRI were used for the experiment each year, but at each site the soil was a free-draining sandy loam, although derived from different soil series and with different cropping histories. Preceding crops in the 1985 site on the Cowpow series were brassicas, spring barley and field beans, whereas in the 1986 site on the Gavock series they were two crops of winter wheat followed by spring barley. MP plants and chitted seed were hand-planted directly into pre-formed potato ridges in the field in May, and each of the treatments comprised a single row of 30 plants 30 cm apart, replicated four times in a random block design. Adjacent treatments were separated by unplanted ridges giving an inter-row distance of 150 cm. Pesticides were applied following commercial practice, but herbicides were used only in a pre-plant soil treatment. Detection of fungi in roots

The fungi present on roots of six MP plants from each treatment were recorded at the time of planting. In later samples roots from six adjacent plants per block from each of the nine treatments in the field were collected in the third week of July, August and September, and processed within 24 h. Root segments were prepared, surface-sterilized in 0.5 % NaOCl and cultured on tap water agar containing streptomycin and chlortetracycline hydroxychloride as previously described (Dashwood ef al., 1992). Twenty segments were selected at random from a pooled sample of segments from the proximal, mid and distal regions of each plant. A total of 120 segments per treatment was assessed at the time of planting, and 480 segments per treatment at each harvest date. The numbers of different species on each segment were recorded and identified where possible, and those not immediately recognized were subcultured, usually on potato dextrose agar, for observation of typical cultural characteristics. Prolonging incubation of the original cultures at 5O or at room temperature sometimes assisted diagnosis of species slow to develop reproductive or resting structures. The isolation medium was not suitable for detecting species of Pyfhiurn because of their sensitivity to the antibiotics used. Statistical analysis

The effect of the nine treatments and three harvest dates on the proportion of infected root segments was analysed separately for selected fungi using frequency data transformed to logits. Interactions between treatment and harvest date and between pairs or groups of treatments were also evaluated for significance. For clarity the frequency of root infection by the

735

E. P. Dashwood, R. A. Fox and J. M. Duncan most commonly occurring fungi is presented unaltered as percentage histograms (Fig. I), but the significance of any treatment or harvest effects stated in the text is based on the analysis of transformed data. A correlation matrix based on a regression analysis for the proportion of roots infected by any two fungi is also presented to indicate positive and negative associations. Spatial relationships between the fungi were determined using principal co-ordinate analysis based on the Jaccard similarity coefficient (Digby & Kempton, 1987). The similarities were calculated by reference to the presence or absence of each fungus in the plant roots.

tabacinum. All others occurred on less than 5 % of the roots, and the majority, comprising about 75% of the species recovered, had a mean frequency of > 1%. In Table 4 the fungi are arranged in rank order of their mean frequency of recovery, their relative positions in 1985 and 1986 being indicated in the last two columns. Many roots yielded 'sterile dark forms' or 'microsclerotial forms', which failed to sporulate in culture but are included because of their relatively high frequency. C. desfrucfans (teleomorph Necfria radicicola Gerlach & Nilsson) colonized the greatest proportion of root segments in both years, with recovery rates of 56 and 47% respectively,

RESULTS

Table 2. Incidence (%) of fungal infection on surface-sterilized root segments from micropropagated plants at the time of planting

Fungi present on the planting material

The fungi recovered from the roots of plants growing in the various potting substrates and from the periderm of the seed tubers prior to planting in the field are listed in Tables 2 and 3. Plants raised in perlite (treatment I, hereafter tr 1, etc.) were virtually free of infection, with 2 and 7 % of the root segments infected respectively in 1985 and 1986. The addition of tuber inoculum (tr 2) increased the proportions contaminated to 13 and 20% respectively. The highest levels of infection occurred in roots from the peat or peat-containing compost (tr 3 , 4 and 5): for these three treatments the mean incidence of infection in the two years was 71 and 83% respectively. Trichodema viride Pers. ex Gray, Penicillium spp. and Chysosporium pannorum (Link) Hughes were frequent in both years. Aureobasidium pullulans (de Bary) Amaud and Phoma leveillei Boerema & Bollen were present only in 1985, and a species of Melanospora occurred only in 1986. Tuber microflora did not contribute noticeably at this early stage to root microflora, but the presence of Polyscyfalum pusfulans (Owen & Wakef.) M. B. Ellis, the skin spot pathogen, confirmed the possibility of contamination by this route (Table 2). Periderm samples from the 'blemished' tuber stock which were used to contaminate the MP plants showed high levels of infection by Colletotrichurn coccodes (Wallr.) Hughes (black dot) and Helminthosporiurn solani Dur. & Mont. (silver scurf), but only moderate infection by Verticillium fricorpus Isaac, Rhizocfonia solani Kiihn (black scurf) and Cylindrocarpon desfrucfans(Zinssm.) Scholten (Table 3). Polyscytalurn pustulans, Gliocladium roseurn Bain, Volutella ciliata Alb. & Schw. ex Fr., C. pannorum, Morfierella spp., Fusarium fabacinum (van Beyma) W. Gams and Dendyphion nanum (Nees ex Gray) Hughes occurred at frequencies between 10 and 20%. Fungi present in roots of plants from the field

The number and frequency of species isolated from surfacesterilized root segments increased overall as the season advanced. The final root samples were collected before haulm senescence whilst the roots were still apparently healthy. A total of 25 920 segments was examined in the two seasons, but of the many different fungi recovered (Table 4) only five species occurred at a mean frequency > 5 % overall. These were C. desfrucfans, C. coccodes, T. viride, Microdochiurn bolleyi (Sprague) de Hoog & Hermanides-Nijhof and F.

Treatment"

Year.. . Infected roots

Zb

7

Trichoderma viride Penicillium spp. Chysosporiumpannorum Cylindrocarpondestluctans Fwarium tabacinum Aureobasidiumpullulans Phoma leveillei Chaetomium spp. P~l~scytalumpustulans Verticillium tricorpus Other species

0 1 0 0 0 1 0 0

3 0 0 0 0 0 0 0

0

0

0 0

0 7

13 20 79 89 56 0 8 0 0 0 0 0 0 2 0 5

0 42 1 1 7 1 8 0 6 1 7 0 I5 0 5 2 1 0 0 3 1 6 2 1

85 78 76

82 30 79 40 6 2 3 0 6 1 6 8 2 3 3 3 1 12 7 10 3 3 10 4 0 1 1 0 I I 0 13 0 7 0 0 0 0 2 0 1 0 1 0 2 0 I 1 0 0 0 0 5 0 1 1 7 2 3 9 3 1 9

Names highlighted in bold type are recognized as 'blemish pathogens' of potato tubers. " For details see Table 1. The combined percentages of the individual fungi are often more than the percentage roots infected, because some roots ~ieldedmore than one fungus.

Table 3. Incidence (%)B of fungal infection on surface-sterilized periderm discs from 'blemished' seed tubers at the t i e of planting in 1986

Colletotrichurn coccodes Helminthosporium solani Verticillium tricorpus Rhizoctonia solani Cylindrocarpon destructans Mortierella spp. Volutella ciliata Gliocladium roseum Chrysosporium pannorum Polyscytalurn pustulans Fusarium tabacinum Dendyphion nanum Fusarium culmorum Fusarium spp. Acremonium strictum Phoma eupyrena Trichoderma viride Geotrichum candidum Doratomyces sternonites Names highlighted in bold type are recognized as 'blemish pathogens' of potato tubers. a No. of periderm discs examined = 100.

Mycoflora of roots of potato plants Table 4. Incidence of fungal species on surface-sterilized root pieces of

Table 4. continued.

potato in 1985 and 1986: percentage of root pieces yielding at least one colony of the specified species

Recovery (%)

Recovery (%)

1985

1986

Rankingn Mean

1985 -

Mean

Cylindrocarpon destructans Colletotrichum coccodes Trichoderma viride Microdochium bolleyi Fwarium tabacinum Fwarium oxysporum Verticillium tricorpus Phoma eupyrena Gliocladium roseum Chysosporium pannorum Verticillium dahliae Aureobasidium pullulans Sterile dark fungi Rhizoctonia solani Phoma leveillei Polyscytalum pustulans Penicillium spp. P. simplicissimum P. expansum P. nigricans P. brevicompactum P. hirsutum Penicillium spp. Verticillium nubilum Dendyphion nanum Volutella ciliata Microsclerotial form Pythium spp. Chaetomium globosum Chaetomium spp Chaetomium elatum Chaetomium spp. Mortierella spp. M . elongata M . hyalina M . minutissima M. alpina M. acrotona M. gamsii Mortierella spp. Paecilomyces carneus Melanospora sp. Rhizopus nigricans Fusarium avenaceum Coniothyrium fuckelii Mucor hiemalis Myxomycete Chaetomium crispatum Fusarium culmorum Cladosporium herbarum Phoma spp. Botytis cinerea Paecilomyces lilacinw Verticillium spp. V. psalliotae V. lecanii V. chlamydosporium

Acremonium spp.

0.11

O.llb

0.11

36

1986 --

-

A. cerealis A. furcatum A . kiliense Acremonium strictum Gliomastix murorum Verticillium lateritrum Doratomyces sfemonites Alternaria alternata Sporothrix sp. Verticillium nigrescens Monodyctis spp. Epicoccum nigrum Helminthosporium solani Monodictys levis Penicillium frequentans Phoma exigua var. exigua Cylindrocarpon spp. Fwarium spp. Rhiwpw stolonifer Coniothyrium cerealis Ulocladium atrum Ascochyta sp. Basidiomycete sp. Humicola grisea Phialophora fastigiatu Rhumnichloridium schulzeri Rhizoctonia spp. Phoma glomerata Trichocladium aspentm Trichocladium opacum Trichoderma spp. Acrospeira levis Aspergillw niger Fwarium equiseti Oidiodendron tenuissimum Pyrenochaeta sp. Tetracladium setigerum Alternaria sp. Papulospora sp. Trichocladium pyriforme Arthrinium sp. Geotrichum cundidum Periconia macrospinosa Phoma putaminum Truncatella truncata Zygorhynchw moelleri Apiosordaria verruculosa Cochliobolw sativw Fwarium poae Monodictys putridinis Preussia funiculatn Trichothecium sp. Unidentified Blank root pieces

5.37 8.86

3.75 9.57

4.56 9.22

Names highlighted in bold type are recognized as 'blemish pathogens' of potato tubers. " An equals sign behind a ranking indicates that more than one fungus had the same ranking. These figures are the combined incidences for the genus.

737

E. P. Dashwood, R. A. Fox and J. M. Duncan and T. viride in 11 and 15 % of the segments respectively. Fusarium oxysporum Schlecht. and Verticillium dahliae Kleb. were frequent in 1985 but rarely recovered in 1986. The potato pathogens R. solani and P. pwfulans each had mean frequencies of about 2%, resulting in relatively high positions on the list, but very few root pieces (0.07%) were infected by H. solani. Effect of harvest date

Root infection by C. desfrucfans, C. coccodes, V. dahliae, Phoma leveillei Boerema & Bollen and G. roseum increased in both years as the season advanced, but for R. solani and Phoma eupyrena Sacc. infection increased only in 1986. C. pannorum and Penicillium spp. showed a consistent seasonal decrease in infection in both years, but T. viride only in 1986. The frequency histograms in Fig. 1 represent the proportion of infected root segments present at each of the three harvest dates for samples from the nine pre-plant treatments. Root infection by other fungi was not affected by harvest date. Effect of seed tuber flora

C. coccodes was found on 75 % of the periderm samples and was subsequently isolated from roots of ST plants at an overall mean frequency twice that of roots of MP plants. Addition of potato peelings to perlite but not to peat-based potting substrates enhanced infection (Fig. 1, 1986). H. solani was only occasionally recovered from root samples despite its high incidence on the tuber surface, and the pathogen is not represented in Fig. 1. Seven root pieces were infected in 1985 and eight pieces in 1986; all originated from ST plants or MP plants raised in inoculated substrates. Root infection by the potato pathogens R. solani and P. pusfulans was also increased in the presence of tuber inoculum. Of the remaining seed tuber flora listed in Table 3, only V. tricorpus and G. roseum were important contributors to root infection. Effect of potting substrate

T. viride and other cellulose- and lignin-degrading fungi originating in the peat potting substrates quickly colonized the root surface of MP plants (treatments 3, 4 and 5, Table 2) and continued to affect the composition of the root flora long after the plants were established in the field (Fig. 1). T.viride was recovered from 39 and 44% of root samples in 1985 and 1986 respectively from MP plants which had been raised initially in peat. In MP plants raised in UC compost and perlite, the corresponding incidences were 18 and 33% and 1.1 and 3 % respectively. In contrast, in the ST plants the figures were 1 and 2 % respectively, and these declined progressively in 1986 but not in 1985 (Fig. 1). Species of Penicillium, mainly P. simplicissimum (Oudem.) Thom and P. expansum Link ex Gray, were moderately frequent on the roots of peat- and compost-grown transplants in 1986 and were recovered from 10 and 21 % of the respective root samples collected in July. By September the incidence had decreased to 2 and 5 % respectively, but remained significantly higher than that of the roots of the 47

same plants raised in perlite, or ST plants harvested on the same date (Fig. 1). In 1985 the frequency of Penicillium species in the roots of transplants and subsequent field samples was low. The mean incidence of C. pannorum and A. pullulans in roots originating from the peat substrates was higher than that of the other treatments in both seasons. C. pannorum showed a similar distribution pattern and relationship with harvest date as T. viride, but the pattern for A. pulluhns was less consistent, and the substrate effect observed in 1986 occurred only in the August and September samples. This fungus was not detected in the same year in pre-plant root samples, suggesting that the source of infection may have been soil-borne. Peat and UC compost potting substrates were also associated with increased levels of root infection by P. leveillei and Melanosporn sp. P. leveillei was detected in all treatment samples in 1985 but showed a marked seasonal increase only in those MP plants originating from the UC compost and inoculated peat. Melanospora sp. was exclusive to the peat and compost treatments in 1986, but its incidence declined rapidly after the first harvest date. The highest incidence of F. tabacinum and V. nubilum Pethybr. occurred on the roots of plants which had been raised in perlite. In 1985 the incidence of these fungi ranged from 1% and < 1% respectively in peat to 9% and 5 % respectively in perlite. In 1986 F. tabacinum was recovered from 28% of samples from the perlite treatments, in contrast to 4 and 2 % of samples from the peat and ST treatments respectively, but the incidence of V. nubilum was low and was not affected by the pre-plant treatments (Fig. I). Effect of field site

The frequent occurrence of the soil-borne fungi F. oxysporum and V. dahline in root samples harvested from the 1985 field site and their absence in corresponding samples from the 1986 site indicated major differences between the two soil populations, possibly related to the different cropping histories. ~ l s o - t h eresults may have been influenced by large differences in the soil moisture deficits in the two years (Table 5). Other ubiquitous soil fungi such as C. destructans, M. bolleyi and P. eupyrena were common to both sites. Associations and interactions of fungi in roots

The frequency with which any two fungi occurred together on the same root system is summarized in the correlation matrix prepared from regression analysis of the 15 most commonly occumng fungi (Table 6). For each harvest date the total number of plants pooled from all treatments was 216, giving D.F. = 214. T. viride was positively associated with C. pannorum, A. pullulans, P. leveillei and C. destructans in 1985, and with C. pannorurn, Penicilliurn spp. and A. pullulans in 1986. The matrix showed T. viride to have a negative correlation with C. coccodes, M. bolleyi, P. eupyrena, G. roseum, F. fabacinum, V. tricorpus, P. pustulans and R. solani in the first year, and with C. coccodes, P. eupyrena, G. roseum, F. tabacinum, V. tricorpus and P. pustulans in the next. Some fungi, e.g. F. oxysporum and V. dahliae, showed few associations. The mycoparasite G. roseum M Y C 97

Mycoflora of roots of potato plants 1985 1 2 abc abc

Infection

1%)

3 abc

4 abc

5 abc

6 abc

7 abc

8 abc

9 abc

1986 1 2 abc abc

3 abc

4 abc

5 abc

6 abc

7 abc

8 abc

i

Penicillium spp.

40

I

0

40

11

..-Ill I,, Ill

i-.

P, levnillei

1

Melanospora sp.

O0

2.- 20 LL

0

V. dahliae

-

I,

I,. I.. I,. .

.-

.

9 abc

E. P. Dashwood, R. A. Fox and J. M. Duncan

20

1

1985 1 2 abc abc P pustulans

20

1

R. solani

Infection (X)

O

200

100

20

1

-.I

-/ ..

3 abc

--

I,.

-

4 abc

8-

5 abc

73 9

6 abc

7 abc

111 ID.

8 abc

9 abc

.II .I.

1986 1 2 abc abc

-

-

3 abc

4 abc

- -1

5 abc

-

6 abc

7 abc

8 abc

9 abc

,, -I. m i

.

1 Cylindrocarpon

1 P. eupyrena

Fig. 1. Incidence and main source of infection of fungi on potato roots at three harvest dates following nine pre-plant treatments in 1985 and 1986. 1-9, pre-plant treatments as listed in Table I. a, b, c, three harvest samples collected in the third week of July, August and September respectively.

Mycoflora of roots of potato plants

740

Table 5. Cumulative soil moisture deficit (mm H,O) calculated for grass ley in 1985 and 1986

Planting Harvest 1 Harvest 2 Harvest 3

56 34

0 103

1

54

0

80

occurred most frequently on roots infected with C. coccodes, P. pusfulans and R. solani, accounting for the apparent positive association with these pathogens, but was itself suppressed by I. viride and associated fungi. There was a negative correlation between C.desfrucfans and C.coccodes in the late samples of 1985, indicating suppression of C. coccodes in roots heavily infected by C. desfrucfans. In the principal co-ordinate analysis each species was considered as a point in a multidimensional space, the distance between each pair of points being related with the respective coefficient of similarity based on presence or absence in the root system. The coordinates of all the points were calculated and plotted with reference to their principal axes; the resultant groupings of fungi obtained for each harvest date in 1985 and 1986 are shown in Fig. 2. The plots for those fungi which regularly occurred together in the six graphs were assigned to four outlined clusters, A, B, C and D. Of these, cluster A, containing T. uiride, C. pannorum, A. pullulans and P. leveillei in 1985 and T. uiride, C. pannorum, A. pullulans and Penicillium spp. in 1986, is the most clearly delimited group. The component fungi occurred abundantly in roots from treatments incorporating peat-based potting media, and their remoteness from other clusters - particularly cluster B which contained the potato pathogens - indicates a low concurrence with other species. In 1985 the cluster grew progressively denser and more remote as the season advanced, corresponding with an overall population increase (Fig. 1). In contrast, the group showed a decline in frequency in 1986, but the expected reversal in pattern, i.e. from dense to diffuse clustering, was not clearly evident owing to the outlier A. pullulans in the graph for the early harvest, H I . Reference to the frequency distribution of this fungus shows it to be an exception to the general trend; it occurred infrequently in the first harvest and then its incidence progressively increased. Cluster B, comprising R. solani, P. pusfulans, G. roseurn and Verficillium nubilum in both years and also including V . tricorpus in 1985, was less well defined than cluster A, having common boundaries with clusters C and D. Except for V . nubilum the fungi occurred together on the surface of the seed tuber source (Table 3). V . tricorpus was infrequent and generally confined to roots of MP plants raised in inoculated perlite in 1985 (tr 2 ) but was widespread in 1986, with a distribution pattem resembling that of F. tabacinum (Fig. I), possibly accounting for its inclusion in the same cluster (C). The absence of R. solani from cluster B in graph H 1, 1986, may have been due to its very low incidence in the early root samples. It is not clear why species of Paecilornyces (mainly P. corneus and P. lilacinus) occurred in the same cluster as did the potato fungi in the second year. The remaining fungi allocated

to clusters C and D are general root-invading saprophytes commonly present in cultivated soils. C. desfrucfans, C. coccodes, M. bolleyi, P. eupyrena and F. fabacinuh in cluster C together comprised the majority of fungi isolated (Table 4). Their populations may have been augmented by tuber-borne inoculum (Table 3). F. oxysporum and V . dahliae, both soilborne species, were frequent in 1985 alone. Fungi in cluster D tended to be less frequent, and showed an erratic distribution sometimes overlapping clusters B and C.

DISCUSSION The fungi isolated from the potato roots were, in the main, forms regarded as typical components of a root-surface mycoflora. Earlier descriptions of potato root populations (Shrivastava & Saksena, 1968; van Emden, 1972) were based on washed roots and are not directly comparable with that found on surface-sterilized pieces. However, most tissueinvading fungi are also common inhabitants of the rhizoplane (Taylor & Parkinson, 1965; Skipp & Christensen, 1981), and surface-sterilization with NaOCl reduced the frequency but not the range of fungi isolated. In some instances the treatment resulted in an increased recovery rate, e.g. for C. desfrucfans, C. coccodes, Paecilomyces sp. and sterile dark forms (Taylor & Parkinson, 1965; Komm & Stevenson, 1978; Skipp & Christensen, 1981). Fungi which were particularly sensitive to NaOCl were Fusariurn spp. (Alabouvette ef al., 1984), Verficillium spp. (Huisman, 1988), Mucor spp., Trichoderma spp., Gliocbdium spp. and some Phoma spp. (Taylor & Parkinson, 1965; Skipp & Christensen, 1981). These fungi may therefore be either over- or under-represented in the species list. The incidence of other fungi was selectively enhanced by some of the experimental treatments; for example, the introduction of peat-based substrate into the soil in three treatments (tr 3-5) increased the frequency of T. uiride, C. pannorum, A. pullulans, Penicilliurn spp. and P. leueillei. The MP plants used in five treatments were likely to be more affected by changing soil moisture deficits than were ST plants, as they had a shallower and less vigorous root system. Consequently they may have been more susceptible to fungal attack, particularly by stress-related fungi such as V. dahliae. This pathogen was isolated from roots of MP plants twice as often as those from ST plants. It was also one of the few common fungi not suppressed by the antagonists associated with the peat amendments. The early widespread infection of roots by C. desfrucfans, which accounted for over 50 % of the fungal isolations in this survey, may have inhibited colonization by other fungi. Van Emden (1972) found similar high frequencies of C. desfrucfans in potato roots, but concluded that it did not prevent colonization by other fungi. The correlation matrix shows negative associations of C. destrucfans with R. solani, C. coccodes, F. tabacinum and G. roseum in 1985, and with F. tabacinum, G. roseum and V . tricorpus in 1986, indicating prior occupation or domination by either of the compared fungi. However, in the frequency histograms the distribution patterns of most of the above fungi fluctuate independently of C. destrucfans, indicating a lack of suppression by the latter. For C. coccodes the pattem was similar, and it would be easy to

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Fig. 2. Spatial relationships of potato root fungi; principal co-ordinate analysis of similarities based on the Jaccard coefficient. Outlined clusters contain species with high concurrence. A, fungi associated with peat-based potting media; B, fungi associated with the seed potato; C and D, common root-invading saprobes. Ap, Aureobasidium pullulans; Cc, Colletotrichum coccodes; Cd, Cylindrocarpon destructans; Cp, Chrysosporium pannorum; C. spp., Chaetomium spp.; DF, dark sterile fungi; Fo, Fusarium oxysporum; Ft, F. tabacinum; Ftcs, Fusarium spp.; Gr, Gliocladium roseum; Mb, Microdochiurn bolleyi; Paec. spp., Paecilomyces spp.; P. spp., Penicillium spp.; Pe, Phoma eupyrena; Pp, Polyscytalum pwtulans; Rs, Rhizoctonia solani; Tv, Trichoderma viride; Vd, Verficillium dahliae; Vn, V. nubilum; Vf, V. tricorpw.

E. P. Dashwood, R. A. Fox and J. M. Duncan attribute its relatively low incidence in the early harvest samples in 1985 to exceptionally high frequencies of C. desfructans in the same year, and conversely its increased incidence in 1986 to lower frequencies of C. destrucfans. Such a comparison between different years is however not valid because of unknown effects of differences in site and microclimate on the interaction. Moderate to extreme differences in the recovery rate for the other common fungi each year may also be associated with differences in the original soil populations, which were unknown, as well as site effects and environmental conditions. Soil temperatures at 1 0 cm depth were broadly similar each year, as were soil textures - a sandy loam and pH (6.8-7.0) but cropping histories were different, and undecomposed barley stubble was present in the soil in 1986. There were contrasting patterns of rainfall in the two years: in 1985 rainfall was high in late summer and exceptionally so in September, but in 1986 the summer was only moderately wet and the autumn dry. The cumulative soil moisture deficits tabulated in Table 5 summarize the differences. Attempts to infer too much regarding the effects of environment on rootsurface colonization by individual fungi for roots growing in natural conditions must be avoided because of the complexity of interacting factors. Thus although many earlier accounts of root mycoflora have interpreted frequency changes in the component species in terms of their differing ecological requirements, no such attempt is made here. Only those interactions which appear to affect colonization by potato fungal pathogens will be examined. C. coccodes, the black dot fungus, is soil- and tuber-borne, and the next most dominant species in the roots after C. destructans. Its frequency early in the season was reduced by organic amendments, particularly in the moist soil conditions of 1985, implying a susceptibility to the peat-associated antagonistic fungi, of which T . viride was the most frequent. The antagonistic effect seemed to disappear late in the season (frequency histogram) despite a strong negative association shown in the correlation matrix. The anomaly could be explained if the two fungi were consistently mutually exclusive in the roots. In culture plates C. coccodes was frequently hyperparasitized and killed by G. roseum, accounting for the positive association in the matrix, but between these species there was no evidence that root-surface populations of C. coccodes were similarly affected. V. dahliae, a cause of early senescence, preferentially infected the roots of MP plants and otherwise showed no treatment effects. Catani & Peterson (1967) found that T. lignortrm (syn. T. viride) appreciably reduced the incidence of V. dahliae in glasshouse-grown maple and egg plants, so the apparent lack of antagonistic effect by treatments supporting high T . viride populations is surprising. The authors also showed biocontrol by G. roseum, not observed here. V. dahliae, along with C. coccodes, F. fabacinum and Strepfomyces, formed a complex described by Scholte et al. (1985), which was associated with yield decrease in potatoes in The Netherlands. Although there was a positive association between V. dahliae and F. fabacinum early in the season in our experiment, that with C. coccodes was negative. Kotcon et al. (1985) studied the fungi involved in the

743 potato early-dying syndrome in Wisconsin and also found a negative association between root infection by V. dahliae and C. coccodes in the presence of, but not the absence of, R. solani, even though the latter was detected only at low levels (2-3%). They concluded that C. coccodes did not predispose plants to infection by V. dahliae. V. fricorpus was considered to be an important biocontrol agent of V. dahliae in potato-growing areas of Idaho and Oregon by Davis & Sorensen (1985). They observed a consistent mutual antagonism between populations of the two fungi, and found positive correlations between V. tricorpus populations and potato yield. Populations of V. fricorpus in the V. dahliae-infested site at SCRI were too low to show such an effect, but it was interesting to note that root populations of V. tricorpus were relatively high in the non-infected 1986 site. Unlike V. dahliae, V. fricorpus was strongly antagonized by the Trichoderma group. Species of Fusarium pathogenic to potato tubers such as F. sobni var. coeruleum (Sacc.) Booth and F. sulphureurn Schlechtendahl were rarely isolated from roots. F. oxysporum was recovered frequently in 1985, and except for early inhibition by the Trichoderma group was unaffected by the treatments. Tivoli (1988) also found this form to be the most common Fusarium sp. in potato roots. We cannot account for its absence in 1986. R. solani, the cause of black scurf, was mainly isolated from roots of ST plants, in which it showed an erratic distribution. Its mean frequency (3.2 %) was low, and similar low frequencies in roots of growing plants were reported by Spencer & Fox (1979) and Kotcon ef al. (1985). The pathogen occurred too infrequently on potato roots in The Netherlands to be included in van Emden's (1972) list of 15 most frequent fungi. R. solani is more sensitive than other soil pathogens to the antagonistic influences of other micro-organisms (Domsch & Gams, 1968; Pugh & van Emden, 1969).Two fungi with known antagonistic activity against R. solani, C. desfrtrctans and T. viride (Domsch & Gams, 1968) also showed negative associations with R. solani in the correlation matrix. T . viride was associated with both low and high black scurf counts in The Netherlands, but mostly low. The high counts were attributed to the suppression of V. biguftafum,a hyperparasite of R. solani, by T. viride (Jager & Velvis, 1989). Mall (1979) noted associated Rhizoctonia resistance in cv. Up-to-Date with high populations of T. viride, and demonstrated direct parasitism in culture plates. Although there are many reports of hyphal penetration of R. solani by T. viride, Griffin (1972) considered mycoparasitism to be a factor of little ecological importance. In a field study he found only six hyphae out of 15000 to be parasitized. G. roseum showed a similar positive association with R. solani as with C. coccodes, perhaps related to its mycoparasitism of R. solani in culture. The skin spot pathogen P. pustulans was isolated from only 2 % of the root segments and was almost entirely tuber-borne. A better recovery rate might have been achieved for this slowgrowing fungus by the use of an appropriate selective medium such as FHC medium (Bannon, 1975). Root infection by P. pustulans is generally accompanied by lesion development and extensive root browning (Hirst & Salt, 1959; Salt, 1964; Hide, 1969), but there have been no previous reports describing its

Mycoflora of roots of potato plants distribution on healthy roots. In the correlation matrix P. pustulans was positively associated with V. fricorpus, F. tabacinurn and G. roseurn, saprobes with which it also co-exists on the tuber surface, and it was negatively associated with T. viride, Penicillium spp., C. pannorum, P. leveillei and A. pulluhns, all fungi associated with the organic potting medium. There was no evidence of the antagonism between R. solani and P. pustubns observed in roots by Spencer (1967) or in tubers by Hide ef al. (1973) and Adams et al. (1980), possibly because of the low frequency of both fungi. These studies did not clearly establish which specific soil- or tuber-borne fungi may reduce root infection by potato pathogens. The complex of micro-organisms in the rhizosphere varies with different soil conditions, climatic factors or cropping history. It includes actinomycetes, bacteria, nematodes and microflora not dealt with here but which directly or indirectly affect the root-surface mycoflora. For example, strong antagonism by Trichoderma spp. may be reduced by Sfrepfomyces spp. and bacteria (Curl, 1973)' and mycophagous microfauna may selectively feed on hyphae of Rhizoctonia (Scholte, 1987). The many uncontrollable factors in this survey may have had greater influence on the fungal interactions than did the imposed treatments. A more systematic approach using controlled environmental conditions may be needed to achieve a better understanding of the behaviour of specific fungi in the field. However, a greater comprehension of the range and frequency of the fungi associated with potato roots under typical field conditions, as attempted to provide by this report, should indicate those areas of interest requiring more detailed epidemiological studies. We thank Dr J. McNicol for help with the statistical analysis, CAB International Mycological Institute Identification Services for confirming identification of many of the fungal species, and the Scottish Office Agriculture and Fisheries Department For financial support.

REFERENCES Adams, M. J. & Hide, G. A. (1980). Relationships between disease levels on seed potatoes, on crops during growth and in stored potatoes. 5. Seed stocks grown at Rothamsted. Potato Research 23, 291-302. Alabouvette, C., Couteaudier, Y. & Louvet, J. (1984). Recherches sur la rbsistance des sois aux maladies. X. Comparaison de la mycoflore colonisant les racines de melons culturbs dans un col rhsistant ou dans un sol sensible aux fusarioses vasculaires. Agronomic 4, 735-740. Bannon, E. (1975). A new medium for the isolation of Oospora pustulans from potato tubers and from soil. Transactions of the British Mycological Society 64,554-556. Blakeman, J.P. & Homby, D. (1966). The persistence of Colletotrichum coccodes and Mycosphaerella ligulicola in soil with special reference to sclerotia and conidia. Transactions of the British Mycological Society 49, 227-240. Carnegie, S. F. & Cameron, A. M. (1990). Occur~ence of Polyscytalum pwtulans. Phoma foveata and Fusarium solani var. coeruleum in field soils in Scotland. Plant Pathology 39, 517-523. Catani, S. C. & Peterson, J. L. (1967). Antagonistic relationships between Verticillium dahliae and fungi isolated from the rhizosphere of Acer platanoides. Phytopathology 5 7 , 363-366. Curl, E. A. (1973). Antagonism in relation to root-mfecting fungi. In The Relation of Soil Microorganisms to Soil-borne Plant Pathogens, pp. 19-23. Research Division of Virginia Polytechnic Institute and State University: Blacksburg, Virginia, U.S.A.

Dashwood, E. P., Fox, R. A. & Peny, D. A. (1992). Effect of inoculum source on root and tuber infection by potato blemish disease fungi. Plant Pathology 41, 215-223. Davis, J. R. & Sorensen, L. H. (1985).Association of Verticillium tricorpw with V. dahliae and early potato dying. Phytopathology 75, 1279. Digby, P. G. N. & Kempton, R. A. (1987). Multivariate Analysis of Ecological Communities. Chapman & Hall: London. Domsch, K. H. & Gams, W. (1968). Die Bedeutung vorfruchtabhangiger verschiebungen in der Bodenmikroflora. 11. Antagonistische Einflusse auf pathogene Bodenpilze. Phytopathologische Zeitschrift 63, 165-176. Fox, R. A. & Dashwood, E. P. (1982). Rhizosphere and allied phenomena affecting plant health. Incidence of selected fungi in potato roots following continuous cropping. Annual Report, Scottish Crop Research Institute 106. Griffon, D. M. (1972). Ecology of Soil Fungi. Chapman & Hall: London. Herzog, W. & Wartenberg, H. (1958). Untersuchungen uber die Lebensdauer der Sklerotien von Rhizoctonia solani (Kuhn) im Boden. Phytopathologische Zeitschrift 33, 291-315. Hide, G. A. (1969). The occurrence, development and control of skin spot (Oospora pwtulans) disease of potato. Ph.D. Thesis, University of London. Hide, G. A. & Adams, M. J. (1980). Relationships between disease levels on seed tubers, on crops during growth and in stored potatoes. 2. Skin spot. Potato Research 23, 215-227. Hirst, J. M. & Salt, G. A. (1959). Oospora pwtulans Owen & Wakefield as a parasite of potato root systems. Transactions of the British Mycological Society 42, 59-66. Hirst, J. M. G., Hide, G. A., Griffith, R. I. & Stedman, 0.J. (1970). Improving the health of seed potatoes. Journal of the Royal Agricultural Society of England 131,87-106. Huisman, 0.C. (1988). Seasonal colonisation of roots of field-grown cotton by Verticillium dahliae and V , tricorpus. Phytopathology 7 8 , 708-716. Jager, G. & Velvis, H. (1989). Dynamics of damage of Rhizoctonia solani in potato fields. In Effects of Crop Rotation on Potato Production in the Temperate Zones (ed. J. Vos, C. D. van Loon & G. J. Bollen), pp. 237-246. Kluwer Academic Press: Dordrecht, The Netherlands. Jellis, G. J. & Taylor, G. S. (1974). Relative importance of silver scurf and black dot; two disfiguring diseases of potato tubers. ADAS Quarterly Review 14, 53-61. Komm, D. A. & Stevenson, W. R. (1978). Tuber-borne infection of Sobnum tuberosum 'Superior' by Colletotrichum coccodes. Plant Disease Reporter 62, 682-687. Kotcon, J. B., Rouse, D. I. & Mitchell, J. E. (1985). Interactions of Vertic~llium dahliae, Colletotrichum coccodes, Rhizoctonia solani and Pratylenchus penetrans in the early dying syndrome of Russet Burbank potatoes. Phytopathology 75, 68-74. Mall, S. (1979). Rhizosphere and rhizoplane microflora of three potato varieties. Indian Phytopathology 32, 51-54. Pugh, G. J. F. & Van Emden, J. H. (1969). Cellulose-decomposing fungi in polder soils and their possible influence on pathogenic fungi. Netherlands Journal of Plant Pathology 7 5 , 287-295. Salt, G. A. (1964). The incidence of Oospora pwtulans on potato plants in different soils. Plant Pathology 13, 155-158. Scholte, K. (1987).The effect of crop rotation and granular nematicides on the incidence of Rhizoctonia solan1 in potato. Potato Research 30, 187-199. Scholte, K., Veenbaas-Rijks, J. W. & Labruykre, R. E. (1985). Potato growing in short rotations and the effect of Streptomyces spp., Colletotrichum coccodes, Fwarium tabacinum and Verticillium dahliae on plant growth and tuber yield. Potato Research 28, 331-348. Shrivastava, K. S. & Saksena, S. B. (1968). Studies on rhizosphere and rhizoplane microflora of potato with special reference to black scurf and wilt diseases. Indian Phytopathological Society Bulletin 4, 107-119. Skipp, R. A. & Christensen, M. J. (1981). Invasion of white clover roots by fungi and other soil microorganisms. I. Surface colonisation and invasion of roots growing in sieved pasture soil in the glasshouse. New ZealandJoumal of Agricultural Research 24, 235-241. Spencer, D. (1967). Some aspects of infection of Solanum tuberosum by Rhizoctonia solani Kuhn. Ph.D. Thesis, University of St Andrews. Spencer, D. & Fox, R. A. (1979). The development of Rhizoctonia solani Kuhn on the underground parts of the potato plant. Potato Research 22, 29-39.

E. P. Dashwood, R. A. Fox and J. M. Duncan Taylor. G. S. & Parkinson, D. (1965). Studies on fungi in the root region. IV. Fungi associated with the roots of Phaseolw vulgaris. Plant and Soil 22, 1-20.

Tivoli. B. (1988). Guide &identification des diffhrentes esphces ou varihths de Fwariurn rencontrees en France sur la pomme de terre et dans son environnement. Agronomic 8, 211-222.

(Accepted 6 November 1992)

745 Tivoli, B., Torres, H. & French, E. R. (1988). Inventory, distribution and aggressivity of species or varieties of Fwarium present in potato or in its environment in different agro-ecological zones in Peru. Potato Research 31, 681-690.

Van Emden, J. H. (1972). Soil microflora in relation to some crop plants. OEPPIEPPO Bulletin No. 7, 17-26.