Introduction

The genus Rhizoctonia was first described in 1815. It is a very diverse group of saprotrophic, pathogenic, and mycorrhizal soil fungi. Pathogenic isolates usually cause damage to underground parts of the plant, but sometimes the disease symptoms are also visible on the aerial parts, or destruction of the whole plant occurs. They can also function perfectly as saprotrophs1. Within this genus, many mycorrhizal species have also been identified, mainly with plants from the Orchidaceae family2,3. The diversity of trophic preferences of isolates of Rhizoctonia spp. causes quality and yield losses in many plant species—cereals, ornamentals, vegetables, trees, and others. Rhizoctonia spp. has been reported in more than 40 countries, but it is possible that they occur worldwide. Population composition is closely related to climate and species composition in a particular area4.

Within the genus Rhizoctonia, the primary classification is based on the number of nuclei in cells and the diameter of hyphae, usually between 3 and 7 µm5. Fungi that have 2 nuclei, the average for this genus, are considered binucleate Rhizoctonia (BNR = binucleate Rhizoctonia) with the Ceratobasidium spp. sexual morph. BNR isolates show pathogenic properties against a wide range of plant species. They can cause various types of damage, such as damping-off and root rot. They may also serve as organisms beneficial to plants, acting as mycorrhizal or protective fungi3,6. Members of the Rhizoctonia genus are basically tested according to thiamine requirement, which leads to indicate some groups as thiamine-auxotrophic or prototrophic. Thiamine plays a stimulative role in auxotrophic organisms, leading to even tenfold growth or greater. Such features were indicated for the members of some anastomosis groups (AG) e.g. R. solani AG-1 or AG-2-2IIIB, AG-2-2IV, and AG-57,8.

Within the same anastomosis groups (AG), there are usually isolates with similar trophic preferences, but the basis for determining group membership is to examine the nature of the reaction between the hyphae of the examined and tester isolates. Molecular analysis of the ITS1-5,8S-ITS2 regions of ribosomal DNA and comparison with the sequence database also allows categorization to the particular AGs and is currently considered the best Rhizoctonia classification method9,10. Classical hyphal fusion is a time-consuming and labor-intensive method. Sometimes its effects are unreliable because some isolates of the same group do not always anastomose or anastomose at a low frequency. It also happens that isolates from different groups anastomose with each other11.

The BNR has been divided into 19 anastomosis groups, from AG-A to AG-W, based on the anastomosis reactions between their hyphae9,10,12. The division into independent anastomosis groups within the BNR group was proposed by Ogoshi et al.13,14 and Burpee et al.15. Ogoshi grouped his isolates into AG-A through AG-Q, while the North American isolates of Burpee were grouped in CAG-1 through CAG-7. Five groups of CAGs turned out to have their counterparts in the AGs proposed by Ogoshi. However, the Ogoshi classification is most often used. The members of the two CAGs were not described by Ogoshi, and they were finally assigned to AG-R (CAG-5) and AG-S (CAG-7). After a more detailed examination, CAG-3 and CAG-6 turned out to be the same group (AG-E), although characteristic anastomosing reactions between them had not been previously observed13. The anastomosis division given by Ogoshi is successfully used up to this day, and it is constantly expanded and modified. Hyakumachi and co-authors16 identified two new, AG-T and AG-U, anastomosis groups within the BNR. Representatives of these groups were isolated from miniature roses grown in Japan. During that time some anastomosis groups were excluded, e.g. AG-J was excluded because it turned out that the fungi belonging to that group formed clamp connections, while AG-M is currently not isolated and does not occur in any of the known collections11,17. AG -T already no longer exists because its isolates anastomosed with AG-A isolates, so the group was included in AG-A11. Analysis of the ITS rDNA sequence of the AG-N group (there is only one sequence available in the NCBI database) showed 61–72% similarity to the other BNR groups, while the sequences of the ITS regions of the other groups are typically similar at the level of 75–95%11. Phylogenetic analysis shows that AG-F, AG-E, AG–P, AG-R, and AG–S arise separately from other BNR clades in dendrograms based on ITS1-5,8S-ITS2 rDNA sequences and are genetically more closely related to R. solani than to BNRs9,18.

However, despite BNRs occurring worldwide, there are not many articles discussing their taxonomically useful features. Hietala et al.19 and Otero et al.2 published uninucleate isolates of Ceratobasidium sp. Moliszewska20 mentioned trinucleate BNRs isolated from sugar beet seedlings’ roots. They were obtained from roots, together with multinucleate R. solani and other BNRs (AG-K). She pointed out that some of the isolates showed a distinct trinucleate state. A piece of similar information was mentioned by Adams and Butler21. They noticed the presence of trinucleate isolates of Rhizoctonia spp. among other isolates. These isolates did not match other Rhizoctonia strains they tested. However, they did not continue their investigation.

According to our observations, the division of Rhizoctonia spp. based on the number of nuclei in binucleate and multinucleate strains may not be obvious. Thus, the aim of this study was to examine a group of previously classified BNR isolates with an unclear two- or three-nuclei stage.

Materials and methods

Sample collection and fungal isolation

The study was based on a collection of fungi of the genus Rhizoctonia isolated from woody and herbaceous plants, as well as sugar beet seedlings from southern Poland (Table 1). The isolates came from the collection of the Department of Forest Ecosystems Protection, the University of Agriculture in Krakow (12 of HS isolates; collection of Hanna Stępniewska Ph.D.) (Table 1). The tester binucleate Rhizoctonia spp. isolates came from the collection of prof. Mitsuro Hyakumachi of Gifu University (Japan), courtesy of Takeshi Toda, Ph.D. of Akita Prefectural University in Akitashi (Japan) (Table 2). Isolation was performed according to classical phytopathological and mycological procedures. The collected research material was stored on PDA slants at 4 °C for immediate use. For long-term storage, cultures were prepared on wheat or millet grains and stored frozen at − 30 °C22.

Table 1 AG-E isolates collected from Poland and used in this study.
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Table 2 Tester isolates of binucleate Rhizoctonia.
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Morphological characteristics, nuclear condition, and hyphae diameter

Isolates listed in Tables 1 and 2 were grown on Potato Dextrose Agar (PDA, Biomaxima) for 2 weeks and then the morphological appearance of mycelium (color and growth intensity), presence of zonation (present/absent), and sclerotia (+—poor, +  +—little, +  +  +—medium, +  +  +  +—abundant) were determined. Cultures that were grown on a PDA medium also were used to evaluate their growth in various temperatures (8, 22, 30 ± 1 °C). Results were shown as daily growth given in centimeters (cm/day). Young Rhizoctonia cultures (3–7 days old) grown on PDA were used to measure the hyphae diameter and number of nuclei per cell. Observations were made microscopically under the 40 × objective, and the diameter of the hyphae was measured on young and well-developed running hyphae, with 50 measurements for each isolate. The number of nuclei in young cells was counted after safranin O staining23 under a light microscope and DAPI (4′,6-diamidino-2-phenylindole) staining under a fluorescent microscope. The mean number of nuclei was determined based on 40 measurements.

DNA Isolation and amplification

Isolates were grown in a liquid peptone medium (per liter: soy peptone—10 g, yeast extract—5 g, NaCl—10 g) for 1 week, after which the mycelium was centrifuged, dried, and stored at − 20 °C for further use. Isolation of genomic DNA was performed using the CTAB method24,25. DNA samples were stored at − 20 °C. Fragments of ITS1-5.8S-ITS2 rDNA were amplified with conventional primers ITS1 and ITS426. PCR reactions consisted of 30 cycles, each with 1 min of denaturation at 93 °C, 1 min of hybridization at 57 °C, and 2 min of elongation at 72 °C. Initial denaturation was conducted for 2.5 min. at 93 °C and the final synthesis lasted for 10 min. at 72 °C (according to27). The products of PCR reactions were purified using an Exo-Sap kit and sequenced at Genomed (Poland). Sequences were compared with those deposited in GenBank using the BLAST tool28.

Determination of anastomosis groups

The initial determination of the AGs was done by comparison with the GenBank sequences. Subsequently, the results were verified according to the classical method of Kronland and Stanghellini29 and Moliszewska and Schneider30 with the testers described in Table 2. The type of reaction was determined according to the criteria given by Carling et al.31; at least 5 contact points showing a C2-type reaction (Fig. 1) were used to confirm AG; observations for each isolate were repeated at least twice. These tests were done for randomly selected isolates—HS27, HS29, HS30, HS38, HS46, HS47.

Figure 1
figure 1

A, B—killing reaction (C2 fusion) between tested isolate and tester and C—self-fusion between hyphae of the same isolate (C3–perfect fusion) (phot. D. Maculewicz).

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Phylogenetic relationships

The phylogenetic relationships of our BNR were visualized using the dendrogram generated based on the ITS1-5.8S-ITS2 sequences. We used our BNRs’ sequences as well as sequences of various BNR anastomosis groups from the GenBank database (Table 3). Other sequences of Rhizoctonia AG-E isolates tested previously by Moliszewska20 were included in this research. The ITS1-5.8S-ITS2 rDNA sequence of Athelia (Sclerotium) rolfsii (isolate FSR052, accession number in GenBank AY684917) was used as an external group (according to11).

Table 3 Reference GenBank sequences used in phylogenetic analysis.
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The BLAST program28 was used to check whether homology existed between the isolates (value limit E > 10–3). Sequence comparison was performed with ClustalW32 and with MEGA X33 and then corrected according to the suggestions of Sharon et al.11. The phylogenetic tree was generated using ITS1-5.8S-ITS2 sequences rDNA with MEGA X33 by the Maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and the Tamura-Nei model34. The tree with the highest log likelihood (− 5080,16) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The proportion of sites where at least 1 unambiguous base is present in at least 1 sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved 50 nucleotide sequences. There were a total of 685 positions in the final dataset. Evolutionary analyses were conducted in MEGA X33. The reliability of the tree was evaluated by the self-sampling method (bootstrap) with 2000 replications.

Thiamine requirement

Thiamine requirement was tested for 8 isolates—HS10, HS23, HS27, HS29, HS30, HS38 and HS47. For this purpose, isolates were cultivated on the pure liquid Czapek-Dox medium (CzD) and liquid Czapek-Dox + thiamine medium (CzD-th) with a thiamine concentration of 10–5 M. The cultivation was carried out for 14 days at 23 ± 1 °C. The mycelium obtained at that time was filtered off, dried, and the dry mass was determined. The auxotrophy was determined according to the ratio of the dry mass of mycelium grown on a medium with the addition of thiamine (CzD-th) to the dry mass of mycelium grown on the medium without the addition of thiamine (CzD). Ratio CzD-th/CzD < 1.5 means prototrophic fungus, while ratio CzD-th/CzD > 1.5—the fungus is auxotrophic to thiamine31. The experiment was performed in four replications.

Statistical analysis

Results were analyzed using ANOVA in Statistica software and Excel (p = 0.05). The significance of differences among results was calculated using the Duncan test (p = 0.05), and standard deviations were also calculated.

Results

Morphological identification, nuclear condition, and hyphae diameter

Tested isolates were primarily accessed to the Rhizoctonia genus according to morphological features and after measurements of hyphae diameter, they were considered BNRs. The hyphae diameter ranged from 3.03 µm for HS21 to 5.27 µm for HS23 (Tab. 4). The average number of nuclei in cells of the test isolates ranged from 1.9 for the HS21 isolate to 3.6 for the HS10 and HS23 isolates (Tab. 4). There was a moderate positive correlation between the diameter of the hyphae and the number of nuclei in cells of tested isolates (correlation coefficient = 0,5088). Using this characteristic, the isolates were divided into two groups. In the first group were fungi which formed cells with only two nuclei (2n group), and in the second group, fungi which showed a varied number of nuclei, usually close to three (3n group) (Fig. 2, Table 4).

Table 4 The average number of nuclei per cell in tested binucleate Rhizoctonia isolates.
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Figure 2
figure 2

Nuclei condition in AG-E cells; pink arrows show nuclei, green arrows show cell walls (HS-30) (phot. E. Moliszewska).

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AG-E isolates grown at room temperature (22 ± 1 °C) presented the majority of the fluffy, creamy, or brown mycelia; a few of them were white; with or without zonation on the culture surface. All of them developed monilioid cells and sclerotia, however, their abundance varied from poor ( +) for HS21, HS29, and HS26 to abundant (+ +  + +) for HS10, HS33, and HS47 (Table 5). There was no regularity and dependency in mycelium colour, its zonation and sclerotia presence according to the number of nuclei if they were considered as two groups (2-nucleic and 3-nucleic isolates).

Table 5 Morphological characteristics of binucleate Rhizoctonia isolates.
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Molecular identification and phylogenetic analysis

The molecular identification of tested isolates was based on the ITS1-ITS2 sequences and included a comparison to other isolates with the BLAST tool (“Supplementary Information”). This showed that our isolates belong to Ceratobasidium sp./Rhizoctonia sp. AG-E. Six of them and one CAG-6 were used as reference sequences in constructing a phylogenetic tree (Table 3, Fig. 3). The observed percentage identity in BLAST was over 99% with E value = 0 for most of our sequences; however, in some cases, e.g. HS29, it was lower, achieving the level 95% of identity to the sequence AB290018. The affiliation to AG-E was randomly confirmed for several isolates, listed in Table 4, by classical anastomosis fusion resulting in a C2 reaction (killing-reaction) when they were paired with tester AG-E isolates Lu-1 and RH155 and failing with other randomly used testers for other AGs (Table 2 and 4, Figs. 1 and 3).

Figure 3
figure 3

A bootstrap tree showing genetic relationships among the AG-E Rhizoctonia isolates based on the internal transcribed spacer sequences, the tree was rooted on the Athelia rolfsii (AY684917) sequence.

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Phylogenetic relationships in BNR

Using phylogenetic analysis, a phylogenetic tree was composed of our isolates, both tested in this research and those listed in Table 7, they belong to both HS and ID groups. They were compared to reference isolates of AG-E and other AGs found in the GeneBank database. The ITS1-5.8S-ITS2 sequences of our isolates clustered with sequences of Ceratobasidium sp. AG-E isolates and one Rhizoctonia sp. AG-E isolate, apart from other anastomosis groups. Two sequences, HS30 and HS38, showed more comparability to a sequence of CAG-6 accession AF354083 (Fig. 2). There is no distinct separation of 2-nucleate and 3-nucleate isolates, which means that 3n isolates are not possible to separate by molecular methods based on ITS1-ITS2 sequence.

Searching accessions in GeneBank showed a broad spectrum of host plants from which isolates were obtained. Among various host plants, there were wheat, carrots, flax, bean, and Erigeron sp. representing weed plants, as opposed to crop plants (Table 4).

Growth rates in different temperatures

The average growth rates of the tested BNR isolates ranged from 1.84 to 2.88 cm per day at 24 °C. The slowest growth was shown by the HS21 isolate, and the fastest by HS47 (Fig. 4). The statistical analyses did not allow separate homogeneous groups (According to Duncan’s test), which means that there were no significant differences among tested isolates in the average daily growth rate in 24 °C; however, the p-value for daily growth was at the level 0,001. Among them, 7 isolates presented the highest growth speed on the second day of growth, while 5 grew the fastest on the third day of development, and only one (HS47) grew the fastest on the first day of cultivation (Fig. 4). Lowering the temperature had a significant effect on the growth rate of the tested isolates. At 8 °C it was much slower than at 24 °C, but it was not completely inhibited—it ranged from 0.31 (HS27) to 0.65 (HS33) cm per day (Fig. 5). Increasing temperature to 30 °C caused increasing the growth rate, however, it was significantly inhibited comparing to the growth rates at 24 °C. The growth rates of BNR at 30 °C ranged from 1.05 cm/day (HS10 isolate) to 1.75 cm/day (HS26 isolate) (Fig. 5). Isolates differed in the growth rates at all tested temperatures, which was confirmed by Duncan’s test analysis. However, it was not possible to separate homogenous groups for the same temperature among tested strains, which shows that they belonged to only one group and behaved similarly.

Figure 4
figure 4

The daily rates of hyphal growth of binucleate Rhizoctonia isolates in 24 °C.

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Figure 5
figure 5

The daily rate of hyphal growth of selected binucleate Rhizoctonia isolates in 8, 24 and 30 °C.

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Thiamine requirement

The test for thiamine requirement in AG-E Rhizoctonia isolates showed that only two of the tested isolates were auxotrophic (HS29 and HS23) (Table 6). There is no possibility to determine any relationship between nuclei number and auxotrophy—this feature seems to exist only occasionally within this Rhizoctonia group.

Table 6 The ability of the tested binucleate Rhizoctonia isolates to synthesize thiamine (auxotrophic isolates are marked in bold).
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Discussion

The results of this research led to a description of a novel feature, based on an average nuclei number in cells, helpful in Rhizoctonia fungi determination. Additionally, tested isolates were described morphologically and microscopically. We did not find any atypical features in the morphology appearance of the group. They presented typical Rhizoctonia genus colors, creamy to brown, and hyphae structure, and they created monilioid cells and sclerotia, however with various abundance. The AG-E consists of both thiamine- auxotrophic and prototrophic isolates. The same was observed by Carling et al.31 in the case of R. solani AG-9, while for other anastomose groups, thiamine requirement is reported as characteristic for the whole group, thus thiamine requirement cannot be used as a diagnostic characteristic for AG-E isolates. The growth rates of tested isolates were quite fast at 24 °C, which seems to be typical for members of the Rhizoctonia genus, while it is slower at lower temperatures as well as at higher temperatures. Our isolates presented diverse sources of isolation, coming from herbaceous to woody plants.

Genus Rhizoctonia includes many species that are morphologically, ecologically, and genetically diverse. Their teleomorphs generally belong to the phylum Basidiomycota, and the class Agaricomycetes. Typically, the term “Rhizoctonia” is understood to mean fungi classified in the genus Rhizoctonia D.C. (syn. = Moniliopsis Ruhland.) with the teleomorph Thanatephorus Donk, for which the basionym is the multinucleate R. solani J.G. Kühn (family Ceratobasidiaceae). The binucleate “Rhizoctonia” (BNR) isolates with teleomorphic stage Ceratobasidium D.P. Rogers were formerly referred to as the taxon Ceratorhiza R.T. Moore. The type species is C. goodyerae-repentis (Constantin & L.M. Dufour) R.T. Moore, and its synonym is Rhizoctonia goodyerae-repentis Constantin & L.M. Dufour (family Ceratobasidiaceae). This large complex of fungi also includes genera that are often called “Rhizoctonia-like”: Chrysorhiza, Thanatophytum, Opadorhiza, Epulorhiza, and Ascorhizoctonia (family Corticiaceae, phylum Ascomycota)6,35. The informal basis for differentiation within Rhizoctonia spp. is the system of anastomosis groups. In the 1930s, Matsumoto et al. and Schultz, independently of each other, observed the phenomenon of anastomosis (fusion) between the hyphae of Rhizoctonia spp. isolates, and their research provided the basis for the currently used classification system for genus5,36. Anastomose groups are denoted by numbers for multinucleated Rhizoctonia spp. or by capital Latin letters for binucleate (BNR) strains. This system is informal, although widely recognized due to its usefulness in classifying and distinguishing individual isolates. Classification within the Rhizoctonia complex is based primarily on anamorphs since obtaining teleomorphs is difficult. Anastomosing reactions between Rhizoctonia spp. hyphae are related to the genetic relatedness of the isolates, but observing anastomose reactions is difficult, requires experience, and is time-consuming and laborious. This is why the comparison of sequences is a frequently chosen method for the determination of AG affiliation. Sharon et al.11 pointed out that the 95–100% sequence similarity and the localization of the ITS1-5.8S-ITS2 sequence on a phylogenetic tree within a cluster of a particular anastomosis group is sufficient to determine the identity of the studied isolate.

We confirmed the AG affiliation, primarily according to the sequence similarity, and then the anastomosis reaction gave us confirmation of our molecular findings. However, morphological observation showed us that on the microscopic level, our isolates differed in the number of nuclei per cell. Some of them distinctly showed three nuclei in the majority of cells. All of them belonged to AG-E. Simultaneously, within the AG-E, considered to be binucleate13,35, apart from the other binucleate AGs, we found isolates containing various numbers of nuclei in cells (from one to four), the average number of nuclei for some of them was about three, and many cells presented a distinctly tri-nucleic state. Moliszewska20 isolated several trinucleate isolates belonging to AG-E, indicating that research in this direction should be continued in order to determine whether this gives the basis for the separation of an individual subgroup of trinucleate Rhizoctonia spp. Furthermore, in the work of Adams and Butler21, there is a mention of the unusual trinucleate Rhizoctonia sp.; however, they did not define their affiliation to AG and did not undertake further considerations on this phenomenon. Our own observations regarding the number of nuclei in AG-E cells clearly indicate that there are both binucleate and trinucleate strains within this group, which can be the basis for dividing this group into two subgroups—binucleate (AG-E-2n) and trinucleate (AG-E-3n). It was also observed that the hyphae diameter of Rhizoctonia spp. isolates is related to the number of nuclei in the cell—larger hyphae diameters were usually found in trinucleate than in binucleate isolates, which was confirmed by the moderate positive correlation coefficient (0,5088) between these two features.

Sharon et al.11 indicated that the commonly accepted division into the bi- and multinucleate mycelia of Rhizoctonia spp. is not so simple, which is also confirmed by our own research. In Scandinavia, uninucleate isolates were found with the Ceratobasidium bicorne teleomorph19,37,38. Uninucleate Rhizoctonia spp. were also isolated by Otero et al.2 from tropical orchids in Central America and by Zhou et al.39 from grasses and maize in China. Hietala et al.19 and Otero et al.2 observed that some isolates formed both uni- and binucleate mycelium. In our collection, there were no uninucleate isolates. Additionally, it is worth mentioning that Ogoshi4 described four-nucleate isolates of R. solani AG-4 as an anamorph of Tanatephorus praticola, which is currently considered R. solani, however, Mordue et al.40 recognized definitely AG-4 as T. praticola, emphasizing it as separate species. Typically it is concluded that isolates containing four or more nuclei are considered R. solani, and those with fewer than four nuclei are concluded BNRs. The results of this research allow the conclusion that the determination of the average number of nuclei in Rhizoctonia spp. cells may be much more useful than previously thought. A typical diagnostic procedure for this type of fungi is microscopic observation (distinguishing characteristic hyphae with branching at almost a right angle and a septum located just behind the branching, lack of clump-connections), followed by measurement of the hyphae diameter and observation of the number of nuclei in the cells. This is the distinction between MNR and BNR. MNRs have wider hyphae (usually greater than 7 µm) and a large number of nuclei (4 to a dozen) in the individual cell. The last two steps are often omitted by researchers, resulting in misclassification. Our own observations suggest the necessity of counting nuclei in actively growing cells as routine management. If it is established that the mean number of nuclei is close to 3, it can be assumed that the isolate belongs to AG-E. Thus, in a simplified way, it is possible to determine the taxonomic affiliation of an isolate to AG-E. However, it should be remembered that within AG-E there are also binucleate isolates, which will not make it possible to negate the belonging to this group for binucleate mycelium and will be a reason for introducing other diagnostic techniques. Moliszewska20 analyzed several isolates of BNR and showed a 3-nuclei (3n) group of isolates giving some additional characteristics. She found that among 3n isolates, there are possibly two zymogram patterns, and simultaneously they also have similar ITS1-ITS2 sequences and were different from the AG-K isolates (Table 7). However, cluster grouping on the dendrogram does not support the observation of two zymogram patterns given by Moliszewska20 (Table 7).

Table 7 Characteristics of bi- and three-nucleate Rhizoctonia spp.; features which were of the same quality were assigned by one or two stars (*, **) (according to Moliszewska20; corrected).
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Rovira et al.41 reported that the ability to synthesize thiamine is a property of the whole anastomose group, not an individual strain. Despite this, fungi of the genus Rhizoctonia may be divided into groups or subgroups based on the ability to synthesize thiamine, but we did not observe such results among AG-E isolates. There were thiamine auxotrophs and autotrophs within this group; however, thiamine auxotrophy seems less frequent than thiamine autotrophy.

Isolates of Rhizoctonia sp. AG-E are able to infect various plant species, including crop plants as well as trees and weeds. Our isolates came partly from trees, like scot pine and silver birch, and partly from crop plants such as lupin, white mustard, vetch, and sugar beets. AG-E testers were isolated from cocksfoot and flax, and searching the GeneBank database brought several other host-plant species such as wheat, carrots, flax, bean, and Erigeron sp. (Tables 1, 2, 3). Those plants belong to various taxonomic groups, thus the AG-E isolates do not show any specific specialty, or on the contrary, they present a very broad specialty against host plants.

Conclusions

The current statement of nuclear conditions possible to observe in the Rhizoctonia genus indicates five possible average numbers of nuclei in cells, namely strains that may be uni-nucleate, binucleate, three-nucleate, four-nucleate, and multiple-nucleate. According to our findings, we propose a simplification in the method for diagnosis of trinucleate strains of the Rhizoctonia-like group. Directly observing the tri-nucleate (3n) condition allows strains to be identified as Rhizoctonia sp. AG-E; however, we strongly suggest also using the sequencing method for confirmation of the AG statement.