fungi associated with strawberry root rot in illinois

Mycopathologia et Mycologia applicata, vol. 41, 3-4 pag. 331-346, 1970

F U N G I A S S O C I A T E D W I T H S T R A W B E R R Y R O O T R O T I N I L L I N O I S 1

by

S. NEMEC e

ABSTRACT

A qualitative and quantitative study of the fungi associated with apparently healthy and root rot-diseased strawberry main roots was made during a I-year period. Eighty-one genera were isolated from lesions and stele segments of diseased roots, and tips and segments 5-6 cm from the tip of apparently healthy roots.

A diverse mycoflora was isolated from each segment of tile root. However, each segment had a typical dominant mycoflora, indicating that a changing mycoflora is associated with the root as it passes from a healthy to a diseased condition. Pythium spp. and Rhizoctonia Spp. accounted for 25.09 and 5.67 percent, respectively, of the isolates from Surecrop lesions, and 4.96 and 25.92 percent, respectively, of the isolates from Cyclone lesions.

INTRODUCTION

Root rot diseases of strawberries have been reported in this coun- t ry for over 50 years (7). During this period strawberry root rots have been reported from Great Britain (3), France (17), Holland (16) Australia (1), and other parts of the world.

Soil-borne fungi, nematodes, bacteria, and winter damage most often have been ment ioned in the many studies concerning the causes of strawberry root rots. Prior to 1950 fungi as potential causes of these root rots were studied more intensively than other factors. These studies revealed tha t a large number of soil fungi are associated with diseased roots. Species of Rhizoctonia (6,36), Fusarium (2,25), Cylindrocarpon (4,34), and Pythium (10,33) were among the fungi consistently isolated from diseased roots. Species of Cylindrocladium (32), Coniothyrium (3, 4, 30), Hainesia (4, 30), and A lternaria (32), were reported to be pathogenic to strawberry roots. Phoma (34), Verticillium (36), Cephalo@orium (31), Stem- phylium (34), Aspergillus (18), Pachybasium (9), and Penicillium (14) were infrequently isolated from diseased roots.

D. C. NORTON (22) reported that by 1950 other investigators had

i Cooperative Investigations, Crops Research Division, Agricultural Research Ser- vice, U.S. Department of Agriculture, and Plant Industries Department, School of Agriculture, Southern Illinois University, Carbondale, Illinois. ~Research Plant Pathologist, Crops Research Service, Agricultural Research Service, U.S. Department of Agriculture, Carbondale, Illinois 62901 Accepted for publication: 10. X. 1969

332 s. ~ c

listed at least 38 fungus genera in their studies of strawberry root rots. In his work species of more than 19 genera were isolated from strawberry roots. In 1962 CHEN (5) reported isolating 3,865 fungus isolates representing 96 genera from lesioned roots. However, only 13 of these fungi were identified to genera or species. As a result of aseptic inoculation studies, 27 fungi were shown to be pathogenic, Despite the large amount of research done on this problem, PLAt<I- DAS (24) stated that 'no positive conclusions can be drawn regarding the causes of root rot.'

The present research was undertaken because of the uncertainty regarding the causes of strawberry root rots. The objective of this s tudy was to identify the fungi isolated from diseased and apparently healthy strawberry main roots and determine their frequency of occurrence duting a 1-year period.

MATERIALS, METHODS, AND EXPERIMENTAL CONDITIONS

Two commercial strawberry plantings, with root rot symptoms, were chosen for this study. A field of Surecrop plants at Dix, Illinois, growing in Bluford silt loam soil was the oldest of the two plantings, having been continuously cropped with this variety for 7 years. Plants in this field were more severely diseased than the Cyclone variety in the second planting near Cobden, Illinois. The Cyclone planting in severely eroded Hosmer silt loam soil was 3 years old.

Runner and mother plants were collected from the two fields in October, 1967 and thereafter at bimonthly intervals through Octo- ber, 1968. Plants were collected from one area of each field, twice each month they were sampled, where they appeared to be uniformly diseased. The fields were not irrigated; therefore, extensive runner plant root growth was delayed until the fall rains began in October and November. Soil temperatures ill both fields remained above freezing until the last week of December.

Root systems were first washed in water. Roots selected to be sectioned were removed from the plant and washed an additional 30 min in running water. Root segments were cut 1--2 mm in length, dipped in 0.26 Yo sQdium hypochlorite solution for 15 sec, and given two 1-minute washes in sterile water.

Four areas of the main root system were selected for this study. Segments were cut from the first cm of the tip, and from the region 5--6 cm (5--6 cm segments) from the tip of apparently healthy runner plant roots. Root lesions were cut from diseased runner plant roots where apparently healthy tissue was becoming necrotic, and stele segments were cut from diseased roots of mother plants of the runner plants. Steles in the lesioned runner roots were too thin to separate easily from the cortex and therefore were not used. Ap- proximately 100 root segments from each part of the main root system were plated on agar from each variety each month, except during April, June and August. In April and June no apparently

FUNGI ASSOCIATED WITH STRA~¥BERRY ROOT ROT 333

healthy Surecrop root segments could be found. No apparently healthy Cyclone segments were found in June, and no 5--6 cm segments were found on Cyclone in August. A total of 4,557 root segments were plated on agar during the year.

All root segments were placed on potato-dextrose agar (Difco), containing 30 ppm rose bengal and 100 ppm streptomycin (PRSA), in polystyrene petri plates. Four segments were placed in each plate. Plates were stored in the dark at 25 C and periodically examined for fungi. Most freely sporulating fungi not requiring special media for identification were identified directly on the plate. Sporulation was attempted with nearly all of the nonsporulating and weakly sporulating fungi (20).

RESULTS

The total number of segments plated on PRSA from the main root system, the percentage of segmen*s that yielded bacteria, fungi or no microorganism growth, and the total number of fungi isolated are given in Table I. A total of 8,102 fungus isolates grew from 2,659 of the 4,557 root segments plated. Bacterial growth developed from 1,009 segments, and no microorganism growth was observed from the remaining 889 segments. A higher percent of fungi grew from lesioned segments of both varieties than from the other three kinds of segments. Tips of both varieties yielded the lowest percent of fungus growth and the highest percent of no growth. Occur- rence of bacteria was highest from the steles of both varieties and lowest from the lesions of the varieties.

A list of the fungi isolated from the root segments is presented in Table II. Data are given as percent frequency of isolation from each root segment category for each variety. Species of 81 genera were isolated from the four root segments of both strawberry varieties. The fungi were grouped into 3 classes and 1 form-class. Identi- fication of the Fungi Imper/~cti was made with taxonomic studies which used the system of classification presented by SACCaRDO (26, 27, 28), and the classification system devised by HUGHES (12).

Lesions of both varieties yielded 8 different fungi and sterile mycetia with isolation frequencies above 3 percent. Pythium spp., Aspergillus niger, Fusarium oxy@orum, F. solani, Trichoderma spp. and Rhizoctonia spp. were isolated from both varieties. Penicillium citrinum series was also isolated from Surecrop lesions and P. purpurogenum series was isolated from Cyclone lesions. Rhizoctonia spp. accounted for 5.67 and 25.92 percent of all isolates which grew from Surecrop and Cyclone lesions, respectively. Pythium spp. accounted for 25.09 percent of all fungi from Surecrop lesions and 4.96 percent of all fungi from Cyclone lesions. In contrast, Pythium spp. repre- sented 0.36 and 0.97 percent of the isolates from Surecrop and Cyclone 5--6 cm segments, respectively. Also, Pythium spp. repre- sented 2.76 and 1.12 percent of the isolates from Cyclone and Sure-

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crop tips, respectively. The identity of certain Pythium spp. was determined from isolates collected twice during the year from lesions. P. sylvaticum CAMPBELL • HENI)RIX was the most frequently occurring species from both varieties. P. perniciosum SEI~BINOW grew from roots of both varieties. In one sample it accounted for 22 percent of the Pythium isolates. P. irregulare BUISMAN grew from 2 of 50 lesions in one sample.

Ten fungi and sterile mycelia were isolated above 3 percent frequencies from apparently healthy tips of both varieties. In this group A. niger, P. /uniculosum, P. purpurogenum series, Alternaria tenuis and sterile mycelia were isolated from both varieties. In addition, Chaetomium /unicola and Trichoderma spp. were isolated from Surecrop tips and Rhizoctonia spp., Coniothyrium sp. and Chaetosphaeronema sp. were isolated from Cyclone tips.

Several species were detected in tile P. purpurogenum series, only P. rubrum STOLL was identified and it was often isolated from both varieties. Sterile mycelia consisted of almost all of the fungi which did not sporulate in a previous study (20), and the isolates which were sterile during early growth on agar but later were overgrown by fungi or other organisms before sporulation occurred. In the previous s tudy (20) a Cytospora sp. and a Cytosporella sp. remained sterile but later sporulated on a modified Alphacel medium (29) in which carboxymethylceltulose was substituted for Alphacel cellulose.

Twelve fungi and sterile mycelia were isolated from 5--6 cm segments of both varieties with frequencies over 3 percent. A. niger, P. /uniculosum, P. purpurogenum series and Chaetosphaeronema sp. were isolated from both varieties. Encountered from this region of the root and not the tip exceeding 3 percent frequencies were Y. solani, Coniothyrium /uckdii and A. /umigatus.

Cephalosporium asperum, P. /uniculosum, Chaetosphaeronema sp., C./uckelii, Rhizoctonia spp., and sterile myeelia were isolated from the steles of both varieties above 3 percent frequencies. Another Co- niothyrium sp., Triehoderma spp., F. solani, F. oxysporum, and Cepha losporium spp. accounted for the other fungi which occurred above 3 percent from Surecrop stele segments. A. niger, Cladosporium herbarum, P, purpurogenum series and Coniothyrium spp. each accounted for over 3 percent of the isolates from Cyclone stele segments.

Isolates listed as Aspergillus spp., Penicillium spp., Vertieillium spp., Fusarium spp., and Paecilomyces spp. were not identified to species for several reasons. Their growth was too restricted and thus prevented their identification or transfer to other media, or the isolates were overgrown by other microorganisms, and in some cases the isolates were not satisfactorily placed into species, series, or groups. The PaeciIomyees sp. isolated from apparently healthy Surecrop roots was originally described as an Acrophialophora sp. Isolates fitting the description of Paehybasium were included in

FUNGI ASSOCIATED WITH STRAV~rBERRY ROOT ROT 343

Trichoderma spp. as recommended by HUGI~ES (13). Rhizoctonia isolates resembling Ceratobasidiurn ROGEI(S (23) were encountered. These and other Rhizoctonia isolates were identified as Rhizoctonia spp. Included in Cephalosporium spp. are C. aeremonium Cda., certain other Cephalosporium isolates, and isolates identified as Hyalopus sp. and Tilaehlidium sp.

D I S C U S S I O N

Nearly all of the fungus genera which have been reported by investigators who have studied strawberry root rot diseases in the United States were isolated from roots of these 2 varieties. Species of 81 genera were isolated from the 4 main root segments of both strawberry varieties. Although the primary reason for this s tudy was to learn the identity of root surface and root inhabiting fungi, association of many of them with different parts of the root may suggest the kind of influence that they have in root rot development.

Of the 45 genera isolated from tips of both varieties 9 occurred with frequencies exceeding 3 percent. The most common of these A. niger and P. ]uniculosum accounted for 13.79 and 13.10 percent, respectively, of the isolates from Surecrop tips. Many of the same genera were consistently isolated from segments 5--6 cm from the tip suggesting that early colonization of the root by these fungi can occur at the tip region. Root rips weakened by factors such as drying conditions or herbicide damage would be exposed to the influence of many different fungi some of which may act as weak pathogens under these circumstances.

A cosmopolitan mycoflora was isolated from each segment of the root. However, each segment had a typical dominant mycoflora indicating that a changing mycoflora is associated with the root as it passes from a healthy to a diseased condition. A. tenuis, F. oxy- sporurn, F. roseum, F. solani, Pythium spp., and Rhizoctonia spp., in general, were associated more with lesions than with 5--6 cm segments, whereas P. ]uniculosum, P. purpurogenum series, Chaeto- sphaeronema sp., C. ]uckelii and a Coniothyriurn sp., in general, were more frequently isolated from 5--6 cm segments than lesions. A. niger, C. asperum, Trichoderma spp., and C. /unicola were isolated from both segments with about the same frequencies. In general, C. asperum, Chaetosphaeronema sp., C. ]uckelii, and two Coniothy- rium species were isolated from the stele more frequently than from lesions or 5---6 cm segments.

Results of microscopic studies (11,22) have indicated a low occurrence of fungi in surface discolored and diseased strawberry root cells. HILDEBRAND & KOCH (11) mainly found Pythium, Rhizoc- tonia, and a mycorrhizai Phycomycete within strawberry roots during a 30-day period after seed germination. From a total of 170 strawberry seedlings they grew in a soil in which field plants became diseased, an average of 10.1 percent showed infection by unidenti-

344 S.~MEC

fled fungi. In these and other similar root disease studies certain mycoflora not considered to be pathogens but which colonize apparently healthy roots may have weakened the root system by pro- ducing toxins or by obtaining access to the root ceils through de- teriorating root hairs or breaks in main roots caused by emerging lateral roots. The contribution to disease development by these root colonizing fungi is difficult to assess. This would be especially true when the root passes into a diseased condition where they are com- peting with other organisms for the substrate. In this regard, Chaeto- @haeronema sp. and Tetracladium marchalianum were isolated from Surecrop 5--6 cm segments with frequencies of 4.73 and 2.91 percent respectively, and from Cyclone 5--6 cm segments with frequencies of 4.18 and 2.89 percent, respectively. T. marchalianum was not isolated from lesions of either variety and the isolation of Chaetosphaeronema sp. from lesions dropped to 0.25 percent fiom Surecrop and 1.42 percent from Cyclone. Because both fungi grow slowly on agar the frequency of their isolation from lesions could have been reduced through competition with faster growing organ- nisms; or their presence could have been altered by microorganism or host metabolites.

C, ARREN (8) found that isolation procedures influenced the apparent make-up of the microflora of peanut pods. In addition, factors such as soil type, soil pH, and the season during which roots are sampled may influence the frequencies with which many organisms are isolated from roots. Therefore, it is difficult to make com- parisons with results of fungus isolations from roots sampled in different strawberry growing regions. However, results of such investigations suggest that fungi which show a specificity for strawberry roots can change, qualitatively and quantitatively, in different strawberry growing regions. Working with Canadian soil HILDEBRAND & KOCH (11) mentioned isolating Thielaviopsis basi- cola (BERK. & BR.) FERR. from roots, a fungus not found from roots in these tests. Idriella lunata, commonly isolated from diseased roots in California (19), was isolated only once from one variety. Also not isolated from these varieties was Diplodia, reported by No- LAN (21) to cause a quickkilling root rot in Florida. These fungi causing damage to strawberries may be limited to certain strawberry growing regions. It is even more difficult to evaluate the participation of nonsporulating fungi in root rot development, within an area, or from region to region.

Many different fungi reside in the strawberry root rhizosphere (15), so that it would not be unusual to isolate many of the fungi that have even a casual association with the root during its development. Organisms of the rhizosphere community by their location and mass possess a distinct advantage over organisms of the general soil microflora when it comes to competition for substrates such as recently dead or senescent rootlets, heartwood of older live roots, and infected roots (35). Collectively, fungi that can cause disease

FUNGI ASSOCIATED WITH STRAWBERRY ROOT ROT 345

symptoms through toxin production or other means, may have a greater potential, during the life cycle of the strawberry root, to cause extensive symptoms than one or two other well-known pathogens. The etiology of root rot development will be better understood when pathogens are identified and when much is learned about how they act on the root.

Zusammenfassung Eine qualitative und quantitative Untersuchung der Prize, die mit den Haupt-

wurzeln yon scheinbar gesunden und Wurzelf~ulnis zeigenden Erdbeeren vergesellschaftet waren, war w~hrend eines Jahres ausgeftihrt. Einundaehtzig Gattungen yon Verletzungen und yon Segmenten erkrankter Wurzeln, wie auch yon Segmenten yon scheinbaren gesunden Wurzeln 5--6 cm yon der Spitze sind isoliert worden. Eine verschiedene Mykoflora wnrde yon j edem Segment der Wurzeln isoliert. J edoch hat te jedes Segment eine typische vorherrschende Mykofiora, die anzeigte, dab eine abwechselnde Mykoflora mit den Wurzeln vergesellschaftet ist, w~hrend sie vom gesunden zum kranken Zustande fibergehen. Pythium spp. und Rhizoctonia spp. waren in 25.0 und 5.6 %, beziehungsweise, yon den Isolaten yon Surecrop L~isionen, und 4.9 nnd 25.9 %, beziehungsweise, yon den Isolaten der Cyclone L~sionen ver- antwortlich.

Acknowledgments The author wishes to thank Dr. W'. A. CAMPBELL, Athens, Georgia for identifying

two Pythium species; Dr L. R. BATRA, Beltsville, Maryland for his evaluation of three genera; Dr. J. J. ELLIS, Peoria, Illinois for identifying the Penicillium and Aspergillus species, and Dr. Paul 2. Nt~LSON, University Park, Pennsylvania for identifying Fusarium solani and F. oxysporum.

References

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fungi associated with strawberry root rot in illinois

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