Zymogram Groups Research Articles

Cross-pathogenicity of Rhizoctonia solani strains on pasture legumes in pasture-crop rotations

In Australia, in the past, pasture legumes were rotated mainly with cereals, but increasingly these rotations now involve pasture legumes with a wider range of crops, including legumes. This increasing frequency of the leguminous host in the rotation system may be associated with increased root rots in legumes in the current pasture-crop rotations. The primary aim of this study was to see whether the pathogenicity on pasture legumes of strains of Rhizoctonia solani sourced from lupins and cereals (common crops in rotation with pastures) is associated with increased incidence of root rots in pasture legumes in the disease conducive sandy soils of the Mediterranean regions of southern Australia. The second aim was to determine sources of resistance among newly introduced pasture legumes to R. solani strains originating from rotational crops as this would reduce the impact of disease in the pasture phase. Fifteen pasture legume genotypes were assessed for their resistance/susceptibility to five different zymogram groups (ZG) of the root rot pathogen R. solani under glasshouse conditions. Of the R. solani groups tested, ZG1–5 and ZG1–4 (both known to be pathogenic on cereals and legumes) overall, caused the most severe root disease across the genotypes tested, significantly more than ZG6 (known to be pathogenic on legumes), in turn significantly >ZG4 (known to be pathogenic on legumes) which in turn was >ZG11 (known to be pathogenic on legumes including tropical species). Overall, Ornithopus sativus Brot. cvs Cadiz and Margurita, Trifolium michelianum Savi. cvs Paradana and Frontier and T. purpureum Loisel. cv. Electro showed a significant level of resistance to root rot caused by R. solani ZG11 (root disease scores ≤1.2 on a 1–3 scale where 3 = maximum disease severity) while O. sativus cvs Cadiz and Erica showed a significant level of resistance to root rot caused by R. solani ZG4 (scores ≤1.2). O. compressus L. cvs Charano and Frontier, O. sativus cv. Erica, and T. purpureum cv. Electro showed some useful resistance to root rot caused by R. solani ZG6 (scores ≤1.8). This is the first time that cvs Cadiz, Electro, Frontier, Margurita and Paradana have been recognised for their levels of resistance to root rot caused by R. solani ZG11; and similarly for cvs Cadiz and Erica against ZG4; and for cvs Charano, Erica, and Electro against ZG6. These genotypes with resistance may also serve as useful sources of resistance in pasture legume breeding programs and also could potentially be exploited directly into areas where other rotation crops are affected by these R. solani strains. None of the tested genotypes showed useful resistance to R. solani ZG1–4 (scores ≥2.0) or ZG1–5 (scores ≥2.5). This study demonstrates the relative potential of the various R. solani ZG strains, and particularly ZG1–4, ZG1–5, ZG4 and ZG6 to attack legume pastures and pose a significant threat to non-pasture crop species susceptible to these strains grown in rotation with these pasture legumes. Significantly, the cross-pathogenicity of these strains could result in the continuous build-up of inoculum of these strains that may seriously affect the productivity eventually of legumes in all rotations. In particular, when choosing pasture legumes as rotation crops, caution needs to be exercised so that the cultivars deployed are those with the best resistance to the R. solani ZGs most likely to be prevalent at the location.

Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia

A collection of isolates of Rhizoctonia solani anastomosis group (AG) 2 was examined for genetic diversity and pathogenicity. Anastomosis reactions classified the majority of isolates into the known subgroups of AG 2-1 and AG 2-2 but the classification of several isolates was ambiguous. Morphological characters were consistent with the species, with no discriminating characters existing between subgroups. Vertical PAGE of pectic enzymes enabled the separation of zymogram group (ZG) 5 and 6 within AG 2-1, but not the separation of ZG 4 and 10 within AG 2-2. PCR analysis using inter-simple sequence repeats (ISSR) and the intron-splice junction (ISJ) region supported the separation of ZG 5 and 6, while the AG 2-2 isolates were separated by geographic region. A comparison of distance matrices produced by the zymogram analysis and PCR indicated a strong correlation between the marker types. Pathogenicity studies suggested canola ( Brassica napus) cultivars were most severely affected by AG 2-1, while cultivars of two species of medic ( Medicago truncatula cv. Caliph and M. littoralis cv. Herald) were susceptible to both AG 2-1 and 2-2. The results indicate that AG 2 is a polyphyletic group in which the classification of subtypes is sometimes difficult. Further investigation of the population structure within Australia is required to determine the extent and origin of the observed diversity.

Intraspecific variation of Rhizoctonia solani AG 3 isolates recovered from potato fields in Central Iran and South Australia

Pectic zymogram, RFLP and PCR analyses were used to characterize Rhizoctonia solani AG 3 isolates collected from diseased potatoes in South Australia. The pectic zymogram data were compared with those obtained for isolates collected from central Iran. Analyses of bands corresponding to pectin esterase and polygalacturonase revealed three zymogram subgroups (ZG) in AG 3. In addition to the previously reported ZG7 (here renamed ZG7-1), two new zymogram subgroups, ZG7-2 and ZG7-3, were identified. Of the 446 isolates tested, 50% of the South Australian and 46% of the Iranian isolates were ZG7-1. The majority of the isolates originating from stem and root cankers were ZG7-1, whereas most of the isolates designated ZG7-2 and ZG7-3 originated from tuber-borne sclerotia. Pathogenicity tests revealed that ZG7-1 generally produced fewer sclerotia and more severe cankers of underground parts of the potato plants than the other two ZGs. Two random DNA clones, one originating from an AG 3 isolate and the other from an AG 4 isolate, were used as probes for RFLP analyses of Australian isolates. The AG 3 probe, previously identified to be specific to this group, detected a high level of genetic diversity, with 11 genotypes identified amongst 50 isolates analysed. The low-copy AG 4 probe resolved three genotypes amongst 24 isolates. For 23 isolates analysed with both markers, the combined data distinguished a total of six genotypes and similarity analysis resolved the isolates into two main groups with 50% homology. PCR, using primers for the plant intron splice junction region (R1), also revealed variation. No obvious relationship among pectic zymogram groups, RFLP and PCR genotypes was observed.

Response of different forms of propagules of Rhizoctonia solani AG2‐1 (ZG5) exposed to the volatiles produced in soil amended with green manures

AbstractThe sensitivity of different forms of propagules of Rhizoctonia solani anastomosis group (AG)2‐1/zymogram group (ZG)5 to volatile compounds produced in soil amended with green manure will influence the efficacy of green manures used to manage the disease. In laboratory experiments, we determined the impact of volatiles arising from residues of five species of Brassicaceae, and Avena sativa (oat), a non‐Brassicaceae species, and volatiles of pure allyl isothiocyanate (AITC) or 2‐phenylethyl isothiocyanate (2‐PEITC) in either their soluble or vapour phase on the hyphal growth of R. solani arising from different propagules. The brassicaceous species were Brassica napus var. Karoo, B. napus B1, B. napus B2, B. nigra and Diplotaxis tenuifolia (a brassicaceous weed). Colony growth and hyphal density on water agar were measured up to 7 days. The amendment of a sandy soil with green manures at a high (100 g kg−1, 10%) concentration generally suppressed the growth of the pathogen, but at a low (10 g kg−1, 1%) concentration, the amendment had little effect on the radial fungal growth of the pathogen but increased the density of hyphae through more branching. The inhibition by volatiles from the residues of Brassicaceae species at 10% concentration was stronger (82–86%) than that by volatiles from oat (64%) amendment at the same rate. Hyphae arising from sclerotia and precolonised ryegrass seed were less sensitive than hyphae growing out of potato dextrose agar plugs. This indicates that thick‐pigmented cell walls may have protected the fungus from these unfavourable conditions. Pure AITC and 2‐PEITC in the range of 0.5–2.0 mM inhibited the growth of R. solani from all forms of propagules, but hyphae originating from agar plugs were the most vulnerable compared with the two others. Thus, hyphae arising from the medulla of the sclerotia may be relatively tolerant to volatile compounds emanating from decomposing Brassica green manure amendments in the field and in vitro inhibition of the vegetative growth of the pathogen may not reflect its response to the amendments in the field.

RAPD-PCR used to confirm that four pectic isozyme (zymogram) groups within the AustralianRhizoctonia solaniAG 8 population are true intraspecific groups

RAPD-PCRs were used to study 79 isolates of Rhizoctonia solani AG 8 representing four pectic isozyme groups [zymogram (ZG)] (ZG1-1, 1-2, 1-4 and 1-5) from various locations in southern Australia. RAPD-PCR was performed on all isolates using six primers and the relatedness of the isolates determined by generating a neighbour joining tree. All isolates were also grouped according to vegetatively compatible populations (VCPs). Within each ZG there were four to nine VCPs. These studies show that the AG 8 population contains four distinct groups that matched the four ZGs and support the concept that ZGs are distinct intraspecific groups.

Pectic zymogram variation and pathogenicity of Rhizoctonia solani AG-4 to bean (Phaseolus vulgaris) isolates in Isfahn, Iran

Rhizoctonia disease, caused by Rhizoctonia solani is one of the most important fungal diseases in bean fields in Isfahan, Iran. Bean plants showing stem and root cankers were collected and Rhizoctonia-like fungi obtained from the samples were identified by anastomosis. Pure cultures of bean isolates of R. solani were identified as AG-4. There were also AG-4 isolates from tomato, potato, cucumber, alfalfa and sugar beet in the areas sampled. A total of 163 isolates of R. solani AG-4 originating from stem and root cankers of beans were examined using pectic zymogram electrophoresis. Polygalacturonase (PG) and pectin estrase isozymes were observed in all AG-4 isolates tested. One (PG) and one pectic esterase (PE) band was found in common between all isolates examined. The electrophoretic patterns were grouped into seven zymogram groups (ZGs) according to the diagnostic PG and PE bands. One ZG occurred in a high frequency throughout the areas sampled. A pathogenicity test was conducted and representative isolates of each ZG were used to inoculate healthy bean plants. The results showed that each ZG caused different symptoms with varying severity. Isolates belonging to two ZGs were highly pathogenic causing root, stem and hypocotyl cankers whereas isolates of the other ZGs produced weak or no symptoms.

Characterization and Pathogenicity of Rhizoctonia Species on Canola.

A total of 112 Rhizoctonia isolates were collected from various canola (Brassica napus) growing areas of Western Australia. Pectic enzyme electrophoresis differentiated these isolates into six distinct zymogram groups: R. solani, 54% ZG5 (AG2-1), 8% ZG6 (AG2-1), and 1% ZG9 (AG10); binucleate Rhizoctonia, 12% CZG1 (CAG1), 4% CZG4, and 6% CZG5 (AGK); and the remainder unidentified binucleate groups (15%). Binucleate groups were also confirmed by fluorescent nuclear staining and hyphal morphology. One or more isolates from each of the above zymogram groups (including four unidentified binucleate groups) and an isolate of ZG1-1 (AG8) that causes bare patch in cereals and legumes were tested for their pathogenicity on canola. Isolates of ZG5 and ZG1-1 were highly pathogenic on canola, delayed seedling emergence, and caused severe hypocotyl and root rot, respectively. ZG5 also induced postemergence damping-off. Increasing the depth of sowing from 1 to 3 cm significantly delayed seedling emergence and increased disease severity. Four unidentified binucleate isolates (WAC9316, WAC9297, WAC9307, and WAC9290) were moderately pathogenic to canola, while two isolates (WAC9307 and WAC9316) caused significant preemergence damping-off. Two CZG5 isolates were weakly pathogenic. Isolates of ZG5 and ZG1-1 were also tested for their pathogenicity on other rotational crops (narrow-leafed lupin, subterranean clover, wheat, oats, barley, and mustard) and two weed species (wild radish and annual ryegrass). ZG5 caused a severe hypocotyl rot on mustard and mild symptoms of hypocotyl rot on narrow-leafed lupin and clover, but failed to infect any of the cereal hosts, such as wheat, oats, barley, and ryegrass. In contrast, all crops tested were highly susceptible to ZG1-1 except mustard, which was only moderately susceptible. Results indicate that ZG5 is most pathogenic to crucifers and is a mildly virulent pathogen of the leguminous crops but not of cereal crops tested. ZG1-1, known to cause bare patch in legumes and cereals, also can cause severe root rot in canola. This is the first report of hypocotyl rot and pathogenicity of ZG5 on canola in Australia.

Relationship of microscopic and macroscopic vegetative reactions in Rhizoctonia solani and the occurrence of vegetatively compatible populations (VCPs) in AG-8

An examination of microscopic and macroscopic vegetative reactions between paired isolated of Rhizoctonia solani AG-8 revealed that pairs of isolates that give a C2 anastomosis reaction at the microscopic level always gives a ‘tuft’ reaction at the macroscopic level. Similarly, any pair that gives a C3 anastomosis reaction at the microscopic level always gives a ‘merge’ reaction at the macroscopic level. Thus, macroscopic vegetative (‘tuft type’) reactions can be used to predict the microscopic (anastomosis) reaction (and vice versa) between paired isolate of AG-8. This pattern also seems to apply to paired isolates of AG-3, AG-7 and AG-10 but not the other AGs of R. solani. The C2 anastomosis reaction is always observed when two isolates from different zymogram groups of R. solani AG-8 are paired. When isolates from within any of the five zymogram groups comprising AG-8 are paired, either a C2 or a C3 anastomosis reaction will result. Many field isolates of AG-8, when paired, give a C3 anastomosis reaction. We propose that a group of field isolates of R. solani that give a C3 anastomosis reaction be designated a ‘vegetatively compatible population’ (VCP).

Identification of Rhizoctonia solani associated with field-grown tulips using ITS rDNA polymorphism and pectic zymograms

Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields. Thirty three AG 2-t isolates, pathogenic to tulips, could be distinguished from AG 1-IC, AG 2-2IIIB and AG 2-2IV, AG 3 and AG 5 by means of ITS rDNA fragment length and after digestion with EcoR I from AG 4 and AG 5. AG 2-t isolates and two Japanese isolates, pathogenic to crucifers and tulips, had an estimated fragment size of 710 bp, whereas Dutch AG 2-1 isolates, non-pathogenic to tulips, showed an estimated fragment size of 705 bp on agarose gel. Digestion of AG 2-t and AG 2-1 isolates with EcoR I, Sau3A I, Hae III and Hinc II revealed four and five distinct ITS rDNA digestion patterns, respectively. In AG 2 isolates 2tR114, 21R14 and 21R61 a double digestion pattern, indicating different ITS sequences within an isolate, was found. The observed ITS fragment length polymorphism between isolates pathogenic and non-pathogenic to tulips were considered too small to be used in routine screening of field isolates. Sequencing of AG 2 isolates 21R01, 21R06, 2tR002 and 2tR144 showed a total ITS rDNA fragment length of 715, 713, 714, and 728 bp. As an alternative to ITS rDNA fragment length polymorphism, pectic enzyme patterns were studied using a commercially available vertical gel-electrophoresis system and non-denaturing polyacrylamide gels amended with pectin. Anastomosis tester isolates AG 1 to AG 11 revealed different ZG. Fifty AG 2-t isolates and five AG 2-1 isolates belonged to a homogeneous pectic zymogram group. We propose to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1. AG 2-1 isolates, non-pathogenic to tulip, formed a heterogeneous group with 4 distinct ZG. Pectic zymography provides an easy, quick and unambiguous method for routine identification of large numbers of field isolates. Such a technique is needed for research on the dynamics of Rhizoctonia populations to develop environmentally friendly control measures of rhizoctonia disease in field-grown flower bulbs.

Characterization of cereal bare patch isolates of Rhizoctonia solani by random amplified polymorphic DNA analysis

RAPD‐PCR was used to characterize isolates of Rhizoctonia solani from bare patches in cereal and pasture crops at two locations in Western Australia, Newdegate and Esperance. All of the isolates belonged to anastomosis group 8, pectic zymogram group 1‐1 or 2. The pectic zymogram assignment could be confirmed by RAPD‐PCR. There was no difference in RAPD‐PCR pattern between highly virulent and weakly virulent isolates at Newdegate, or between isolates from different patches at Newdegate. The Newdegate isolates were identical to isolates from Esperance, and to isolates from various locations in South Australia.

Characterization of a unique population of Fusarium oxysporum f.sp. cubense causing Fusarium wilt in Cavendish bananas at Carnarvon, Western Australia

A unique population of Fusarium oxysporum f. sp. cubense affecting Cavendish cv. Williams banana plants was characterized using vegetative compatibility, volatile production, RAPD-PCR analysis, pectic enzyme production and pathogenicity. The isolates were more like race 1 isolates than race 4 isolates, although they were capable of attacking Cavendish clones. The Carnarvon isolates did not belong to any of the vegetative compatibility groups (VCGs) known to occur in Australia or overseas; they belonged in the 'inodoraturn' volatile group; they had 29% genetic similarity to race 4 isolates and 76% similarity to race 1 isolates based on RAPD-PCR banding patterns; they belonged in the same pectic zymogram group as race 1 isolates and were virulent on 3-month-old Cavendish cv. Williams, Gros Michel and Pisang Gajih Merah plants in glasshouse tests.

Anastomosis group (AG) affinity of pectic isozyme (zymogram) groups (ZG) of Rhizoctonia solani from the Western Australian cereal-belt

Over 4250 isolates of R. solani obtained from the cereal growing belt of Western Australia have been characterized using pectic isozyme patterns (zymograms). With few exceptions, all can be placed in one or other of 10 zymogram groups (ZG), four of which (ZG1-1, ZG1-2, ZG1-4, and ZG1-5) have previously been identified as members of anastomosis group AG-8 and cause rhizoctonia bare patch disease of cereals, legumes and forage pastures. Another (ZG5) has been identified as AG-2-1. The latter identification is confirmed in this study and the anastomosis group affinity of the remaining five ZGs reported. ZG4 and ZG10 are placed in AG-2-2, ZG6 in AG-2-1 and ZG9 in AG-10. ZG3, although it reacts in a very limited way with some isolates of AG-2-1 and AG-8, appears to represent an as yet unreported AG. The potential value of zymograms as an aid to disease management and as a research tool for identifying sub-groups within AGs is discussed. Integration of the AG and ZG systems, and use of ZGs to subdivide AGs is also discussed.

Association of Rhizoctonia strains with bare patch disease of wheat in Western Australia

Rhizoctonia‐like fungi were isolated from the roots of diseased wheat plants sampled from the centre and periphery of three bare patches, and from apparently healthy plants from outside the patches. Of the isolates recovered, 81% were multinucleate and belonged to R. solani anastomosis group 8, and pectic zymogram group 1‐1; the remaining isolates were binucleate Rhizoctonia spp. The multinucleate isolates could be grouped into highly virulent, intermediately virulent, and weakly virulent types. The binucleate isolates were all non‐pathogenic. The multinucleate isolates were obtained at a significantly higher frequency from plants within the patches compared with outside the patches, and with the exception of a single isolate, the highly virulent isolates were not found outside the patches. The weakly virulent isolates were present at much lower frequencies than the highly virulent and intermediately virulent forms within the patches. The frequency of occurrence of binucleate isolates did not vary significantly among the locations sampled. None of the multinucleate isolates contained plasmids. Some of the isolates contained a prominent single dsRNA species and one or more minor dsRNA species. The distribution of these dsRNAs was not correlated with pathogenicity.

Genetic Variability in Pectic Enzymes of Rhizoctonia solani Isolates Causing Bare‐patch Disease of Cereals

AbstractField isolates of Rhizoctonia solani obtained from three discrete bare patches in a wheat field in Western Australia were characterized by pectic zymogram grouping. The genetic background of pectic enzymes was analysed by comparing the zymograms of asexual homokaryons and sexual progenies derived from field isolates. The 170 field isolates obtained from the field site produced indistinguishable pectic zymograms. However, variations among field isolates of the same zymogram group were detected, on the basis of zymograms of their resultant protoplast‐regenerated cultures. Asexual sibling homokaryons derived from each of the field isolates were heterogeneous for their pectic enzymes. Homokaryons with a common heterokary on incompatibility factor, obtained from a field isolates were homogeneous for pectic enzymes. Basidiospore progenies of a field isolate segregated widely in pectic zymograms. It appeared that the expression of pectic enzymes by field isolates involved multiple genetic factors. The variation of zymograms among homokaryotic strains suggests that each field isolate of R. solani contains two types of nuclei, although cells of vegetative hyphae are multinucleate.

Molecular markers in Australian isolates of Rhizoctonia solani

Isolates of Rhizctonia solani from different locations within Australia and Japan were analysed for restriction fragment length polymorphisms (RFLPs). These isolates belong to different anastomosis and pectic zymogram groups. Southern blots of restriction enzyme digested DNA were probed with a cloned 18S ribosomal RNA (rRNA) gene, or random cloned fragments of R. solani DNA. The patterns obtained with the rRNA probe revealed significant variation within some of the groups whereas other groups were more uniform. Group specific patterns could not be identified for all groups. There was less variation in the RFLP patterns when random cloned fragments of DNA were used as probes and group specific patterns could be identified for all groups.

Evidence of stability of pectic zymogram groups within Rhizoctonia solani AG-8

Within R. solani AG-8, the cause of rhizoctonia bare patch disease of cereals, legumes and pasture, five zymogram groups have been identified. Evidence is presented that demonstrates the five groups (currently designated ZG1-1, ZG1-2, ZG1-3, ZG1-4 and ZG2) are very stable. These groups have a wide distribution in the cereal growing regions of southern Australia and some have been detected in the Pacific Northwest of the U.S.A. We propose that ZG2 be renumbered as ZG1-5 to create a standard numbering system for zymogram groups within AG-8.

Analysis of variation in isolates of Rhizoctonia solani by random amplified polymorphic DNA assay

Genetic variation in Australian isolates of Rhizoctonia solani was analyzed by random amplified polymorphic DNA (RAPD) assay. Isolates originated from different geographic locations in Australia and belong to a number of different anastomosis and pectic zymogram groups. Using four different oligonucleotide primers fingerprint patterns were generated for each isolate. All of the anastomosis and pectic zymogram groups (including subgroups) tested could be distinguished. For some groups there was considerable variation in the fingerprint patterns between isolates. This variation was more marked between isolates from different geographic locations. Other groups showed very little variation between isolates. RAPD-PCR analysis is a very useful alternative to anastomosis grouping for identification of isolates of R. solani.

Evidence that each Rhizoctonia bare patch is dominated by an individual zymogram group (ZG) of Rhizoctonia solani AG-8

Studies of rhizoctonia bare patches in the southern part of the cereal belt of Western Australia indicate that each patch is dominated by an individual pectic zymogram group (ZG) of R. solani AG-8. R. solani was isolated from plants sampled from patches or from wheat seedlings grown in undisturbed soil cores removed from patches. The Rhizoctonia spp. isolated were characterized using electrophoresis in pectin-acrylamide gels. Four patch-forming zymogram groups (ZG1-1, ZG1-2. ZG1-4 and ZG1-5) were isolated. Of 121 patches examined, only five yielded more than one ZG. Two of these were due to the coalescing of two patches dominated by different zymogram groups. For the remaining three, there was only an occasional isolation of a ZG different from the dominant ZG. This may indicate a new infection focus endeavouring to establish in soil that another ZG occupies. Occasionally, patch-forming ZG1-1, ZG1-2, ZG1-4 and ZG1-5 were isolated from non-patch sites.

Characterization ofRhizoctonia solaniAG-8 from Bare Patches by Pectic Isozyme (Zymogram) and Anastomosis Techniques

Isolates of Rhizoctonia solani AG-8 collected from Rhizoctonia bare patches at two locations in Western Australia were characterized by pectic isozyme (zymogram) and anastomosis techniques. All isolates collected from the same patch at the same time and at different times up to 10 mo apart were members of the same zymogram group. Anastomosis techniques demonstrated that these multiple isolates from the same patch were members of the same clone, as indicated by the clonal anastomosis reaction. Anastomosis reactions also indicated that isolates from the same zymogram group but from different patches in the same field or farm may or may not be members of the same clone. In general, patches with the same zymogram group and in close proximity to one another are more likely to be of the same clone than are patches separated by greater distances [...]

The use of zymogram and anastomosis techniques to follow the expansion and demise of two coalescing bare patches caused by Rhizoctonia solani AG-8

Pectic zymogram characterization and anastomosis techniques were used to study the distribution of strains of R. solani AG-8 in a large rhizoctonia bare patch. Undisturbed soil cores were removed on nine occasions during the period 1988 to 1992. Identification of the isolates of R. solani from wheat seedlings grown in the cores demonstrated that the patch was a coalescence of two patches, each colonized by isolates of R. solani AG-8 representing different zymogram groups (ZG1-1 and ZG1-5). The demarcation between the two patches remained unchanged while both patches were active. Between the 1989 and 1990 seasons, the pathogen in half of the ZG1-5 patch died, or was reduced to such low levels that it was difficult to detect. In the remainder of the ZG1-5 patch, the pathogen died or was reduced to undetectable levels between the 1990-91 seasons. The ZG1-1 patch was evident at the beginning of the 1991 growing season and the pathogen could be isolated; however, by the end of the season the pathogen could not be isolated. In all these instances the demise of the patch was associated with the disappearance of field symptoms and an inability to isolate the pathogen and therefore the apparent death of the fungus in the soil.