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Biological characteristics and metabolic phenotypes of different anastomosis groups of Rhizoctonia solani strains

BMC Microbiology volume 24, Article number: 217 (2024) Cite this article

Abstract

Background

Rhizoctonia solani is an important plant pathogen worldwide, and causes serious tobacco target spot in tobacco in the last five years. This research studied the biological characteristics of four different anastomosis groups strains (AG-3, AG-5, AG-6, AG-1-IB) of R. solani from tobacco. Using metabolic phenotype technology analyzed the metabolic phenotype differences of these strains.

Results

The results showed that the suitable temperature for mycelial growth of four anastomosis group strains were from 20 to 30oC, and for sclerotia formation were from 20 to 25oC. Under different lighting conditions, R. solani AG-6 strains produced the most sclerotium, followed by R. solani AG-3, R. solani AG-5 and R. solani AG-1-IB. All strains had strong oligotrophic survivability, and can grow on water agar medium without any nitrutions. They exhibited three types of sclerotia distribution form, including dispersed type (R. solani AG-5 and AG-6), peripheral type (R. solani AG-1-IB), and central type (R. solani AG-3). They all presented different pathogenicities in tobacco leaves, with the most virulent was noted by R. solani AG-6, followed by R. solani AG-5 and AG-1-IB, finally was R. solani AG-3. R. solani AG-1-IB strains firstly present symptom after inoculation. Metabolic fingerprints of four anastomosis groups were different to each other. R. solani AG-3, AG-6, AG-5 and AG-1-IB strains efficiently metabolized 88, 94, 71 and 92 carbon substrates, respectively. Nitrogen substrates of amino acids and peptides were the significant utilization patterns for R. solani AG-3. R. solani AG-3 and AG-6 showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 8% sodium lactate. Four anastomosis groups all showed active metabolism in environments with pH values from 4 to 6 and exhibited decarboxylase activities.

Conclusions

The biological characteristics of different anastomosis group strains varies, and there were significant differences in the metabolic phenotype characteristics of different anastomosis group strains towards carbon source, nitrogen source, pH, and osmotic pressure.

Highlights

Phenotyping differences in carbon, nitrogen, and pH metabolism, as well in osmotic pressure among four anastomosis groups (AG-3, AG-5, AG-6, AG-1-IB) of R. solani were different.

Nitrogen substrates of amino acids and peptides were the main metabolic substrates for R. solani AG-3.

R. solani AG-3 and AG-6 showed a wide range of adaptability.

Peer Review reports

Background

Tobacco (Nicotiana tabacum L.) is a major commercial crops in China, with an annual planting area of up to 1 million hectares. China produces nearly 40% of the total global tobacco leaves and 40% of the global tobacco consumption [1]. Tobacco target spot is a devastating disease on tobacco production. It can occur from the seedling stage to the mature stage, while mainly harming tobacco leaves in the field [2]. At the early stage, the disease spots are circular water stains, and the tobacco leaves fade with yellow halos. Laterly, the disease spots expand and form the disease spot with a diameter of 2–3 cm. Typical symptoms of tobacco target spots firstly appear on the old leaves as round watery spots, the tobacco leaves are chlorotic, and with yellow halo [3]. The necrotic parts of the disease spots are fragile, resembling cavities left on the target after gunshot. When humidity rises, the edge of the lesion often produces the mycelium of its pathogen, and future occur fruiting layer and basidiospores of the sexual generation [4]. Then the multiple lesions are connected into patches and leading to perforation and rupture of the leaves, thereby depriving the leaf of its economic value [5].

The pathogen of tobacco target spot is Rhizoctonia solani Kühn. R. solani has affected more than 260 important cash crops, such as potatoes, tomatoes, sugar, corn, wheat and peanuts [6,7,8,9]. In recent years, tobacco target spot has occurred in Guizhou, Chongqing, Yunnan, Sichuan and other major tobacco producing areas [10, 11]. The disease incidence rate of tobacco plants can reach more than 80%, and even more 100% [12], reduce the value of tobacco leaves. R. solani belongs to the Hyphomycetes, Agonomycetales, Agonomycetaceae and Rhizoctonia [13]. It does not produce conidium, and its sexual generation is Thanatephorus cucumber (Frank) Donk [14]. The genetic differentiation of R. solani is complex and its life history is relatively unique [13], and a phenomenon commonly occurring in filamentous fungi has been pointed out as hyphal anastomosis, which is characterized by the exchange of genetic material [15]. In a case study, it has been reported that the mycelial anastomosis phenomenon taxa of R. solani and established the system of mycelial anastomosis group (anastomosis group, AG for short) [16]. The existence of 14 mycelial anastomosis groups of R. solani has been reported, including AG-1 to AG-13 and AG-B1 [17]. Ogoshi further subdivided the anastomosis group into 18 subgroups of R. solani based on the anastomosis group [18]. The R. solani AG-3, which was first identified and reported to have the widest distribution range on tobacco in China [19]. In another study identified the anastomosis group of tobacco target spot pathogens in some tobacco areas of Hunan Province and found that tobacco target spot pathogens belong to R. solani AG-3 [20]. In a recent study identified the anastomosis group of tobacco target spot pathogens in Hubei Province, China, and found that tobacco target spot pathogens belong to R. solani AG-3 [21]. Other anastomosing groups of R. solani have been reported in most tobacco areas of China. Chen et al. identified the anastomosis group of tobacco target spot pathogens in Guangxi Province and found that the pathogens belong to R. solani AG-2 and R. solani AG-4 [22]. Our laboratory identified the anastomosis group of R. solani in tobacco regions of Guizhou and Sichuan provinces in the early stage, which belongs to R. solani AG-5 and R. solani AG-6. This is also the first report of tobacco target spot caused by R. solani AG-5 and R. solani AG-6 on tobacco in China [23, 24].

Biolog metabolic phenotype technology is one of the important methods for studying microbial metabolic function. It is a technology invented by Bochner in the United States in 2000 for measuring cell phenotype [25, 26]. The system can measure nearly 1000 metabolic phenotypes of microorganisms, and can be used in conjunction with computer software for data analysis. It has the characteristics of high automation and standardization, and fast identification speed [27]. It has microporous plates such as GEN III microplates, ECO metabolic plates, FF microplates, and Phenotype Microarray (PM) microplates. Its principle is that during the metabolic process of microbial cells, the free electrons generated by the metabolic carbon / nitrogen substrate undergo a reduction color reaction and turbidity difference with tetrazole dyes [26]. By utilizing a unique phenotypic arrangement technique, the metabolic fingerprint of each microorganism can be detected [28]. This technique can also be used to study the metabolic function of environmental microbial populations. In many studies, it has been used to analyze the activity of microbial communities, or to study the pathogenic mechanism of pathogens and the action mechanism of fungicides through metabolic conditions [28,29,30,31]. In tobacco, Wang et al. used ECO metabolic plates to research the differences in metabolic function of tobacco brown spot pathogen [30]. Similarly, Liu et al. used the ECO metabolic plates to study the metabolic function of microorganisms in the rhizosphere of tobacco leaves with different maturity levels susceptible to brown spot disease [32]. In another study, Wang et al. used Biolog FF microplates to determine the biological activity of azoxystrobin, and salicyloximic acid against Fusarium oxysporum strain from tobacco [1]. In a recent study, Liu et al. used the PM 9–10 microplates to study the metabolic phenotype on different osmotic pressure and pH environments of tobacco black shank pathogen [33]. Previous researchers have conducted in-depth and systematic studies on tobacco brown spot pathogen, black shank pathogen, and powdery mildew pathogen using this metabolic phenotype technology. Nevertheless, there have no reports on the use of metabolic phenotype technology to study tobacco target spot pathogen, and the biological characteristics of different anastomosis groups strains have not reported.

Therefore, the research measured the biological characteristics of different anastomosis group strains, and measured the metabolic phenotypic characteristics of different anastomosis group mycelial to carbon substrate, nitrogen substrate, pH and osmotic pressure using the Biolog metabolic phenotype technology. Determine the pathogenicity of tobacco target spot pathogens in different anastomosis groups on K326 tobacco leaves. The objective of this research was to (i) identify biological characteristics of four anastomosis groups of R. solani and (ii) characterize the metabolic phenotype of four anastomosis groups of R. solani. The data provided by this study will be valuable to expanding the knowledge of the biochemistry and metabolic phenomics of R. solani strains and will ideally assist in the development of more effective measures for tobacco target spot and tobacco sore shin management.

Results

Effect of different temperatures on mycelial growth and sclerotium formation of R. solani at different anastomosis groups

The temperature range is 10℃-35℃ of different anastomosis group strains’ mycelium can grow. The mycelium of four anastomosis group strains can’t grow at too low (5℃) or too high (40℃) temperature, the mycelium grew fastest at 15 and 25℃, followed by 20 and 30℃ (Table 1; Fig. 1). The results of variance analysis showed that there were significant differences between the colony diameters of different anastomosis group strains at the same temperature. The mycelium of R. solani AG-1-IB strains grew fastest at 10, 15, 20, 25 and 30 °C compared to other anastomosis group strains and there were significant differences. The mycelium of R. solani AG-6 strains grew fastest at 35 °C, followed by R. solani AG-5 and R. solani AG-1-IB, and finally the R. solani AG-3. There were significant differences in colony diameters among the four anastomosis group strains.

Table 1 Effects of temperature on mycelium growth of different anastomosis group of Rhizoctonia solani strains
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Fig. 1
figure 1

Colony morphology of different anastomosis group strains of Rhizoctonia solani at different temperatures

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The sclerotium production of different anastomosis group strains was affected by different temperatures, and both low and high temperatures could affect the sclerotium production. Four anastomosis group strains can’t produce sclerotium at 5, 10, 30, 35 and 40 °C, but the R. solani AG-1-IB anastomosis group produced sclerotium after 240 h of cultivation at 15 °C (Table 2). There was no significant difference between the numbers of sclerotium by the three strains of R. solani AG-1-IB. All strains produced sclerotium at 20 °C. The first one produced sclerotium was R. solani AG-1-IB, followed by R. solani AG-3 and R. solani AG-5, and finally was R. solani AG-6, and the sclerotium formation time were 216 h, 264 h, 408 h, 456 h. There were significant differences between the numbers of sclerotium by different anastomosis group strains. All strains produced sclerotium at 25℃, and the first to produce sclerotium was R. solani AG-1-IB, followed by R. solani AG-3 and R. solani AG-5, and finally was R. solani AG-6, and the sclerotium formation time was 168 h, 216 h, 360 h and 408 h. There were significant differences in the number of sclerotium produced by R. solani AG-5 and R. solani AG-6 strains at 20℃. There were significant differences in the number of sclerotium produced by R. solani AG-1-IB and R. solani AG-5, R. solani AG-6 strains at 25℃. However, there was no significant difference in the number of sclerotium produced by R. solani AG-1-IB and R. solani AG-3 strains at 25℃ (Table 2).

Table 2 Effects of temperature on sclerotium formation of different anastomosis group of Rhizoctonia solani strains
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Effects of different light times on the mycelial growth and sclerotium formation of R. solani at different anastomosis groups

The R. solani AG-3 strains grew fastest under 12 h of alternating light and dark conditions, followed by total darkness conditions, and finally was continuous illumination conditions (Fig. 2). The mycelial growth of R. solani AG-5 strains and the R. solani AG-3 was exactly the opposite, with continuous illumination being the fastest condition for mycelium growth, followed by 12 h of alternating light and dark, and finally was total darkness. The fastest growth condition for the mycelium of R. solani AG-6 and R. solani AG-1-IB was continuous illumination, followed by total darkness, and finally was 12 h of alternating light and dark. There were significant differences in the mycelial growth of different anastomosis group strains under the same lighting conditions. Under continuous illumination conditions, the mycelial growth rates of R. solani AG-3 strains were the slowest. There were significant differences in the mycelium growth rates between R. solani AG-1-IB strains and R. solani AG-3, R. solani AG-5, R. solani AG-6 strains under continuous illumination. Under total darkness conditions and 12 h of alternating light and dark conditions, there was significant difference in the mycelial growth rate between R. solani AG-1-IB strains and R. solani AG-3, R. solani AG-5, R. solani AG-6 strains (Table 3).

Fig. 2
figure 2

Colony morphology of different anastomosis group strains of Rhizoctonia solani under different illumination conditions. Note L represents continuous illumination, D represents total darkness, LD represents 12 h of alternating light and dark conditions

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The time required for sclerotium formation of different anastomosis group strains varies under different lighting conditions (Table 3). Under continuous illumination conditions, the R. solani AG-1-IB strains first produced sclerotium, followed by R. solani AG-6 strains, R. solani AG-5 strains, and finally was R. solani AG-3 strains. The four anastomosis group strains required 96 h, 192 h, 312 h, and 408 h to form sclerotia, respectively. There was no significant difference in the number of sclerotia produced by the four anastomosis group strains under continuous illumination conditions. Under total darkness conditions, the R. solani AG-1-IB strains first produced sclerotium, followed by R. solani AG-6 strains, R. solani AG-5 strains, and finally was the R. solani AG-3 atrains. The four anastomosis group strains required 144 h, 216 h, 312 h, and 432 h to form sclerotia, respectively. There were significant differences in the number of sclerotium produced by the four anastomosis group strains under total darkness conditions, and there were significant differences in the number of sclerotium among different strains of the same anastomosis group.

Table 3 Effects of illumination on mycelium growth and sclerotium formation of different anastomosis group of Rhizoctonia solani strains
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Effect of oligotrophic medium on the mycelial growth and sclerotium formation of R. solani at different anastomosis groups

The mycelium of different anastomosis group strains can grow on oligotrophic medium (water agar medium), but the growth rate varies, and the R. solani AG-1-IB strains had the fastest growth rate (Fig. 3; Table 4). The significant differences were observed between colony diameters of strains of different anastomosis groups, while significant differences were observed between strains AG-31 and strains AG-32, strains AG-33 of R. solani AG-3, and between colony diameters of three strains of R. solani AG-5 and R. solani AG-6. The oligotrophic medium inhibited the formation of sclerotium in the different anastomosis groups, and all strains of R. solani AG-3, R. solani AG-5 and R. solani AG-6 can’t form sclerotium during the 50 d observation period, while only the R. solani AG-1-IB strains formed sclerotium, but the number of sclerotium was very small (Fig. 3; Table 4).

Fig. 3
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Colony morphology of different anastomosis group strains of Rhizoctonia solani on water agar medium

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Table 4 Effect of water agar medium on mycelial growth and sclerotia formation of different anastomosis group of Rhizoctonia solani strains
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Analysis of differences in sclerotium formation of R. solani at different anastomosis groups

The four anastomosis group strains can be divided into three types based on the distribution form of sclerotia, including dispersed type, peripheral type, and central type (Fig. 4). The sclerotium distribution pattern of R. solani AG-3 was central type. The sclerotium distribution pattern of R. solani AG-5 and R. solani AG-6 were dispersed type. The sclerotium distribution pattern of R. solani AG-1-IB was peripheral type, and during the sclerotia formation stage, it is observed that some mycelial will grow along the edge of the culture dish and eventually form sclerotia, as shown by the red arrow in Fig. 4. During the observation period, the R. solani AG-1-IB strains had the highest number of sclerotium, followed by R. solani AG-3 strains, finally was the R. solani AG-5 strains and R. solani AG-6 strains (Table 5). The R. solani AG-1-IB strains required the shortest time for sclerotium formation, followed by R. solani AG-3 strains and R. solani AG-6 strains, finally was the R. solani AG-5 strains. In terms of the sclerotium number, the R. solani AG-1-IB strains can form the most sclerotium. There were significant differences in the number of sclerotium between the R. solani AG-1-IB strains and the R. solani AG-5 strains, the R. solani AG-6 strains. However, the number of sclerotium formed by R. solani AG-1-IB strains was higher than that the R. solani AG-3 strains, and there was no significant difference at the sclerotium number between R. solani AG-1-IB strains and R. solani AG-3 strains. There was no significant difference in the sclerotium number between the R. solani AG-5 and the R. solani AG-6 strains (Table 5).

Fig. 4
figure 4

Sclerotium morphology of different anastomosis group strains of Rhizoctonia solani. Note The red arrow in the figure represents the mycelium growing along the edge of the culture dish and eventually forming sclerotium

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Table 5 Time and quantity of sclerotia formation of different anastomosis group of Rhizoctonia solani strains
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Differences in pathogenicity of R. solani at different anastomosis groups on K326 tobacco leaf

Daily observations revealed the leaves inoculated with the R. solani AG-1-IB strains were the first to develop lesion, followed by R. solani AG-5 strains and R. solani AG-6 strains, and finally was R. solani AG-3 strains. The largest lesion diameter at 9 d of disease lesion was on leaves inoculated with the R. solani AG-6 strains, followed by leaves inoculated with the R. solani AG-5 strains and leaves inoculated with the R. solani AG-1-IB strains, and finally was leaves inoculated with the R. solani AG-3 strains (Fig. 5). The R. solani AG-3 strains colonized on leaves and exhibited the light symptoms. The R. solani AG-5, R. solani AG-6 and R. solani AG-1-IB strains colonized on leaves and exhibited extremely severe symptoms. On the 9th day of disease lesion, the diseased leaves of each anastomosis group strains showed severe damage (Fig. 5A). The pathogenicity of the R. solani AG-5, R. solani AG-6, and R. solani AG-1-IB strains was significantly different from that of the R. solani AG-3 strains at 3 d of disease lesion. The pathogenicity of the R. solani AG-6 strains was significantly different from that of the R. solani AG-3 strains at 3, 7 and 9 d of disease lesion (Fig. 5B). In summary, the pathogenicity of different anastomosis group strains differed on the main tobacco varieties. The R. solani AG-1-IB strains being the first to colonize the leaves and show symptoms, and the R. solani AG-6 strains being the severe pathogenic (Fig. 5).

Fig. 5
figure 5

Pathogenicity of different anastomosis groups of Rhizoctonia solani on K326 tobacco

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Phenotype differences in carbon substrate metabolism among different anastomosis groups of R. solani

The research found that the strains from different anastomosis group can metabolize all carbon substrates in the FF microplates. The R. solani AG-1-IB strains had a strong metabolic ability towards other carbon substrates, in addition to a weaker metabolic ability towards D-Galacturonic Acid and Sebacic Acid. The R. solani AG-6 strains can metabolize 95 carbon substrates and had a strong ability to metabolize carbon substrates. The R. solani AG-5 strains had a strongest ability to metabolize α-D-Glucose-1-Phosphate, D-Mannitol, γ-Hydroxybutyric Acid, L-Phenylalanine, while had a weaker ability to metabolize other 91 carbons. The R. solani AG-3 strains can metabolize 95 carbon substrates, among which the utilization ability of D-Galactiuronic Acid, L-Aspartic Acid, L-Fucose, D-Glucosamine, Bromosuccinic Acid, Sebacic Acid, L-Pyroglutamic Acid, 2-Aminoethanol, Putrescine, Adenosine was weak (Fig. 6).

Fig. 6
figure 6

Heat map of 95 carbon sources metabolism abundance of different anastomosis group strains of Rhizoctonia solani Note The legend of colour code from blue to green, and red shades indicate low, moderate, and high utilization of carbon sources, respectively, assessed as arbitrary Omnilog values

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Phenotype differences in nitrogen substrate metabolism among different anastomosis groups of R. solani

The research found that the strains from different anastomosis group can metabolize all nitrogen substrates in the PM 5 and PM 6 microplates with different metabolic capacities. Nitrogen substrates that were efficiently metabolized by the R. solani AG-3 strains include Ile-Arg, Ile-Tyr, Glu-Trp, Glu-Val, Asp-Trp, IIe-Gln and Ala-Tyr. The R. solani AG-5 strains can efficiently metabolize a few substrates such as lle-Trp and IIe-Tyr, and unable to metabolize L-Aspartic acid, Ile-Ser, Gly-Trp. The R. solani AG-6 strains was able to metabolize all nitrogens and can be metabolized efficiently include lle-Trp, His-Trp, Ala-His, His-Tyr, Ala-Arg, Cys-Gly, Ala-Pro, Ala-Asn, Leu-Phe, Gly-Trp, Ala-Gly, Arg-Tyr, Glu-Tyr, Gly-Cys, Ala-Glu, Glu-Trp, while the other substrates have low metabolizing ability. The R. solani AG-1-IB strains can efficiently metabolize His-Tyr, Glu-Trp, Ala-Pro, Ile-Tyr and Glu-Tyr, and can metabolize other substrates with low metabolic rate except for lle-Phe (Fig. 7, Figure S1).

Fig. 7
figure 7

Heat map nitrogen sources metabolism abundance of different anastomosis group strains of Rhizoctonia solani

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Phenotype differences in osmotic pressure and Ph metabolism among different anastomosis groups of R. solani

The strains of four anastomosis groups had weak adaptability to Osmotic pressure and pH environment, among which R. solani AG-3 strains has the most extensive adaptability to pH. At pH = 4.5, it can grow in an environment where 22 substances, including L-Alanine, L-Arginine, L-Asparagine, and L-Aspartic Acid, coexist respectively. It can grow in the environment of Sodium sulfate 2%, Sodium sulfate 3%, Sodium sulfate 4%, Ethylene glycol 10%, Ethylene glycol 15%, Ethylene glycol 20%. The R. solani AG-5 strains can only grow under four kinds of Osmotic pressure, namely, Social formate 1%, Social formate 2%, Social Lactate 1%, and Social Nickel 10mM. It can’t grow in five kinds of environments where pH and nutrients coexist, namely, pH 4.5 + L-Leucine, pH 4.5 + Anthranilicacid, pH 4.5 + L-Norleucine, pH 4.5 + p-Aminobenzoate, pH 9.5 + Phythylamine. The R. solani AG-6 can grow under 21 kinds of Osmotic pressure, including NaCl 2%, Sodium formate 1%, Sodium formate 2%, and Sodium Lactate 1%, but cannot grow under pH 4.5 + Anthranilicacid. The R. solani AG-1-IB can grow in all pH or pH coexisting environments with nutrients, but has a low metabolic rate of nutrients (Fig. 8, Figure S2).

Fig. 8
figure 8

Heat map of pH and osmotic pressure metabolism abundance of different anastomosis group strains of Rhizoctonia solani

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Discussion

The Rhizoctonia solani is a destructive fungal pathogen distributed worldwide. Many molecular biological, genetic and genomic studies have been conducted on R. solani [34, 35]. Although this pathogen is commonly found in tobacco, potato, rice, wheat and cucumber hosts, the biological characteristics and metabolic phenotypic diversity of R. solani and its different anastomosis groups is still poorly understood [36]. The Biolog FF system and Biolog PM system have received considerable attention in population studies of many microorganisms [37]. This study not only studied the biological characteristics of four anastomosis groups of R. solani strains, but also the metabolic ability of four anastomosis groups of R. solani strains obtained from tobacco was systematically studied using Biolog FF microplates, Biolog PM microplates and important metabolic diversity information was obtained. The data obtained in this study on R. solani and its optimal growth conditions, metabolic functional diversity, pathogenic differences can play a very significant role in developing prevention technologies for leaf spot caused by R. solani on tobacco.

The R. solani can infect more than 200 plant species worldwide [38, 39]. Different anastomosis groups can infect different crops, such as R. solani AG-3, which mainly infects tobacco and potato [40,41,42,43,44]. At the same time, in a recent study revealed that R. solani AG-5 and R. solani AG-6 can infect tobacco [24, 25]. From previous work, it has been demonstrated that different crops have different nutrition substrates, different osmolytes and pH environments in their tissues, which affect the survival and pathogenicity of pathogens [45, 46]. The hosts of R. solani are differ in their taxonomy, and the biological characteristics metabolic phenotypic characterization of R. solani strains also differ greatly.

Temperature and light have a significant effect on the biological characteristics of R. solani. The results of this study showed that the four R. solani anastomosis groups stranis can grow at 10 –35 °C, and 20 –25 °C was suitable for the growth of the sclerotium, which was basically consistent with the result of Wu et al. [14]. When the temperature exceeded 25℃, the mycelial of R. solani growth was slow, and when the temperature exceeded 35℃, the mycelial of R. solani growth stop. Based on this result, temperature control measures can be taken in tobacco seedlings to prevent mycelium of R. solani from colonizing the seedlings and to reduce seedling diseases. This result can provide more measures for the prevention and control of tobacco target spot. For example, in tobacco production, when the soil temperature reaches 25℃, it can be used as the first application time to prevent and control tobacco target spot. At the same time, before and after the tobacco plant enters the bud stage, the soil temperature can be reduced by opening the plastic film to reduce the occurrence of the disease. Illumination has an impact on the mycelial growth and sclerotium formation of R. solani. This study found that under the condition of 12 h of alternating light and dark, the R. solani AG-3 strains had the fastest mycelial growth. Under continuous illumination conditions, the R. solani AG-6 strains and R. solani AG-1-IB strains had the fastest mycelial growth. Under total darkness conditions, the R. solani AG-1-IB strains had the fastest mycelial growth. The above results were consistent with the research results of Sneh et al. [47]. The mycelial growth and sclerotium formation of R. solani were significantly affected by temperature and illumination, and suitable temperature and illumination are conducive to R. solani mycelial growth and sclerotium formation. Therefore, it can be effectively prevented and controlled according to the biological characteristics of the pathogen. Indeed, illumination conditions control measures can be taken in tobacco seedlings to prevent mycelium of R. solani from colonizing the seedlings and to reduce seedling diseases. In addition, this study also found differences in the pathogenicity of different anastomosis groups strains, which was consistent with the research results of Zou et al. [20]. It is speculated that the expression levels of pathogenic genes in the four anastomosis groups strains are inconsistent, leading to differences in pathogenicity. This hypothesis needs to be verified in the next study.

The metabolic phenotype characteristics of microorganisms under different osmotic pressures and pH can reflect their adaptability to the environment. The metabolic phenotypes of different R. solani anastomosis group strains on carbon substrate, nitrogen substrate, pH and Osmotic pressure were different. Carbon substrate is the basic nutrient for biological survival, and nitrogen substrate can provide the nitrogen required for the growth and development of organisms [32]. Studies have shown that microorganisms can utilize nutrients in FF microplates and PM microplates, as reported by Wang et al. [48]. Streptomyces can metabolize 77 carbon substances in Biolog FF plates. Wang et al. reported that Kyushu Fusarium can efficiently metabolize 69 carbon substrates in the FF microplates and can moderately metabolize 18 carbon substrates [49]. This article found that the R. solani strains of four anastomosis groups can metabolize all carbon substrates in FF microplates, indicating that R.solani has a stronger ability to metabolize carbon substrates compared to other pathogen. Reducing the addition of carbon sources during seedling cultivation to inhibit the growth of R. solani. From previous work, it has been demonstrated that tobacco brown spot pathogen can efficiently metabolize over 60 nitrogen substrates, including L-glutamic acid and L-lysine, in PM3 microplates [50]. The R. solani studied in this article can utilize 190 nitrogen substrates, and the nitrogen substrates that can be efficiently metabolized include L-glutamic acid. In addition, this study found that R. solani AG-3 strains has stronger Osmotic pressure and pH environment adaptability than other anastomosis groups strains, which is conducive to the R. solani AG-3 to survive many adverse environments. It could also be one of the reasons why R. solani AG-3 strains are more common than other anastomosis groups strains. R. solani AG-3 has a wide range of adaptation to pH, indicating that acidic conditions are more favorable to its growth than alkaline conditions, and it should be a weakly acidophilic pathogen. Therefore, in production, attention should be paid to soil acidity and alkalinity as well as the previous crop planted when planting tobacco in order to minimize the occurrence of diseases. The number of carbon substrates metabolized was highest for the R. solani AG-6 strains from tobacco target spot leaves. The reason for this difference is unclear and might be that R. solani AG-6 strains contains more genes for metabolizing carbon substrates than the other anastomosis groups strains. More work could be conducted to verify this hypothesis in the next study. Different concentrations of osmotic pressure substances can be applied to control tobacco target spot disease or reduce the degree of harm caused by the disease, which requires further experiments to determine. In addition, the growth environment of tobacco target spot pathogen is complex in the field, and further research on the pathogenicity factors of tobacco target spot pathogen is needed under various factors such as soil type, microorganisms, and host resistance interaction modes in the future.

Materials and methods

Origin of R. solani strains

The tested strains in different anastomosis groups were identified and stored at Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science. Three strains (J215, J216, J136) of the R. solani AG-6 and three strains (B6-8, B7-1, T1-141) of the R. solani AG-5 were the first identified new anastomosis groups on tobacco in Guizhou, China. Three strains (AG-31, AG-32, AG-33) of the R. solani AG-3. Three strains (LK1, LK2, LK3) of the R. solani AG-1-IB were selected as control, which causing tobacco sore shin. The strains information is shown in Table S1.

Test materials and instruments

The test medium is potato dextrose agar medium (PDA: 200 g of potato, 17 g of agar powder, 20 g of dextrose, 1000 mL of distilled water, autoclaved). Water agar medium (oligotrophic medium, WA: 20 g agar powder, 1000 mL distilled water, autoclaved). Potato dextrose broth medium (PDB: potato 200 g, glucose 20 g, distilled water 1000 mL, autoclaved). The tested tobacco seedlings are K326 tobacco seedlings with good growth and healthy appearance. The reagents and instruments were Biolog FF microplates, Biolog PM microplates, FF-IF inoculum, OmniLog system and 8-channel electric pipette, all purchased from Biolog, U.S.A. D-glucose (Sigma, U.S.A.), yeast nitrogen substrate (Difco, U.S.A.).

Effect of temperatures to mycelial growth and sclerotium formation of R. solani

The test strains were cultured on PDA medium for 3 d, and the PDA plugs with mycelial were made at the edge of the colonies with a sterilized punch with an inner diameter of 6 mm, inoculated into the middle of PDA medium, and incubated in the dark incubators at 5, 10, 15, 20, 25, 30, and 35℃, respectively. Four replicates for each treatment and after 48 h of incubation, the colony diameters were measured. The measured plates were continued to be incubated and observed, and the status of the colony plates was observed daily, and the number of sclerotium was recorded at the beginning of sclerotium formation and 10 d after sclerotium formation.

Effects of light condition to mycelial growth and sclerotium formation of R. solani

The test strains were cultured on PDA medium for 3 d, and the PDA plugs with mycelial were made at the edge of the colonies with a sterilized punch with an inner diameter of 6 mm, inoculated into the middle of PDA medium. The test plates were incubated at 25 °C, continuous illumination, 25 °C, complete darkness and 25 °C, 12 h alternating light and dark, respectively. Four replicates were conducted for each treatment, and after 48 h of incubation, the colony diameters were measured. The method for observing the number of sclerotium formation was the same as above.

Effect of medium to mycelial growth and sclerotium formation of R. solani

The test strains were cultured on PDA medium for 3 d. The PDA plugs with mycelial was made at the edge of the colony with a sterilized punch with an inner diameter of 6 mm and inoculated into the middle of water agar medium (WA), and incubated in the dark incubators at 25 °C. Four replicates were set up for each treatment, and the colony diameters were measured after 48 h of incubation. We observed the status of colony plates every day, and took photos and recorded at the beginning of sclerotium formation, at the growth stage of sclerotium, at the maturity stage of sclerotium, and finally recorded the number of sclerotium.

Analysis of differences in sclerotium formation of R. solani at different anastomosis groups

The test strains were cultured on PDA medium for 3 d. The PDA plugs with mycelial was made at the edge of the colony with a sterilized punch with an inner diameter of 6 mm and inoculate into the middle of the PDA medium, and cultivate them in dark incubators at 25℃. Four replicates were set for each treatment, observe the status of the colony plates every day, take photos at the beginning of the formation of the sclerotia, record the growth period of the sclerotia, take photos at the mature stage of the sclerotia, and finally record the number of sclerotia.

Pathogenicity of different R. solani anastomosis groups

The test strain was incubated on PDA medium for 3 d. The PDA plugs was made at the edge of the colony with a sterilized punch with an inner diameter of 6 mm (standby). The leaves were collected from the 2nd to 3rd leaf position (from the bottom to up) of the 7 ~ 8 leaf stage tobacco plant of K326 variety. The leaves disinfected with 75% alcohol, washed with sterile water in turn, and air-dried. The same wounds were prepared with sterile inoculation needles at symmetrical sites away from the leaf veins. The wounds were inoculated with 6 mm diameter PDA plugs with R. solani. and the mycelial surface close to the leaf, and each strain was repeated six times. After inoculation, these leaves were placed in an artificial climate incubator (temperature 28 °C, relative humidity 70%, 12 h of alternating light and dark conditions) and incubated for 24 h. The PDA plugs were removed with a sterile toothpick and the inoculated leaves was observed daily. The time of incidence was recorded, and the incidence of leaves was observed at 3 d, 5 d, 7 d and 9 d after the incidence of each anastomosis group of inoculated leaves, respectively. The diameter of each lesion was measured. The differences in the pathogenicity of each anastomosis group strain were evaluated by the size of the lesion after the inoculation of each anastomosis group strains.

Phenotype differences in carbon substrate metabolism among different anastomosis groups of R. solani

The test strains AG-32, B6-8, J136 and LK3 (one from each anastomosis group) were randomly selected from the above study. The test strains (AG-32, B6-8, J136, LK3) were incubated on PDA medium for 3 d. The PDA plugs with mycelia was made at the edge of the colony with a sterilized punch with an inner diameter of 6 mm. The mycelial suspension was made by reference to the method of Zang et al. [51]. The PDA plugs with mycelial was inoculated into sterilized triangular flasks containing PDB liquid medium, and the triangular flasks were placed in a shaker at 25 °C, 180 rpm shaking for more than 96 h. A sufficient amount of mycelium grows in the triangular flask, the solid medium at the time of inoculation was removed with sterilized toothpicks and forceps, and the remaining pure mycelium was filtered and washed with distilled water until the distilled water was clear. The appropriate amount of cleaned mycelium was transferred to a sterile 2 mL centrifuge tube. Adding the appropriate amount of FF inoculum in 2 mL centrifuge tube. The mycelium was ground into uniform mycelial fragments using a grinder at 2000 rpm. All mycelial fragments were transferred to a sterilized triangular flask and FF inoculum was added to make a mycelial suspension. The concentration of the mycelial suspension was adjusted to 62% T (T is the standard concentration unit of Biolog) [52]. The mixed mycelial suspension was added to the Biolog FF microplates using an 8-channel electric pipette at 100 µL per well. The FF microplates were incubated in an OmniLog incubator at 25 °C for 7 d. Established OmniLog working software and collected data. Using DataAnalysis software to analyze data, convert data to. csv format, and further convert data to. xlsx format. The carbon substrate metabolic phenotypic characteristics of the mycelium were analyzed according to its metabolic profile. Useing HemI (version 1.0.3.3) software to plot the heat map.

Phenotype differences in nitrogen substrate metabolism among different anastomosis groups of R. solani

Prepare mycelium suspension according to the above method and adjust the concentration of the mycelium suspension to 62% T. Adding the mixed mycelial suspension to PM 5, 6 microplates using an 8-channel electric pipette at 100 µ L per well. Incubate PM 5, 6 microplates in OmniLog incubator at 25 °C for 7 d. Established OmniLog working software and collected data. Using DataAnalysis software to analyze data, convert data to. csv format, and further convert data to. xlsx format. The nitrogen substrate metabolism phenotype was characterized according to the metabolic profile of the mycelium. Useing HemI (version 1.0.3.3) software to plot the heat map.

Phenotype differences in osmotic pressure and Ph metabolism among different anastomosis groups of R. solani

Prepare mycelium suspension according to the above method and adjust the concentration of the mycelium suspension to 62% T. The mixed mycelial suspension and PM microplates additive [26] were added to PM 9 and PM 10 microplates using an 8-channel electric pipette at 100 µL per well. The PM 9 and 10 microplates were incubated in an OmniLog incubator at 25 °C for 7 d. The OmniLog working software was set up and data were collected. Using DataAnalysis software to analyze data, convert data to. csv format, and further convert data to. xlsx format. The pH and osmotic pressure metabolic phenotypic characteristics of the mycelium were analyzed according to its metabolic profile. Useing HemI (version 1.0.3.3) software to plot the heat map.

Data availability

Data are contained within the article and supplementary materials.

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Acknowledgements

We thank Meili Sun for her assistance in the experimental setup and data collection . We thank Hangcheng Wang , Songbai Zhang , Feng Wang, Liuti Cai, Zhen Li and Guo Ye for their assistance in the manuscript modification . All authors agree to publish the manuscript on BMC Microbiology.

Funding

This research was funded by the National Natural Science Foundation of China (award nos. 32160522 and 31960550), Guizhou Province Applied Technology Research and Development Funding Post-subsidy Project, Guizhou Science Technology Foundation (ZK-2021-Key036, Qiankehe Talent Platform – CXTD[2023]021), China National Tobacco Corporation (110202101048(LS-08), 110202201019(LS-03), 110202001035(LS-04)), Hundred’ Level Innovative Talent Foundation of Guizhou Province (GCC[2022]028 − 1, GCC[2023]108), and Guizhou Tobacco Company Project (nos. 2020XM03, 2020XM22 and 2024XM06).

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  1. MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River (Co-construction by Ministry and Province), Yangtze University, Jingzhou, Hubei, 434025, People’s Republic of China

    Meili Sun, Guo Ye, Songbai Zhang & Zhen Li

  2. Guizhou Provincial Academician Workstation of Microbiology and Health, Guizhou Academy of Tobacco Science, Guiyang, 550081, P. R. China

    Meili Sun, Hancheng Wang, Liuti Cai & Feng Wang

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Contributions

Meili Sun, Hancheng Wang Liuti Cai, Feng Wang contributed to conception and design of the study. Meili Sun, Guo Ye, Zhen Li conducted the experiment and analyzed the data. Meili Sun wrote the main manuscript text and prepared Figs. 1, 2, 3, 4, 5, 6, 7 and 8; Tables 1, 2, 3, 4 and 5, supplementary materia. Hancheng Wang, Feng Wang, Songbai Zhang, Liuti Cai revised the manuscript. All authors reviewed the manuscript.

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Sun, M., Wang, H., Ye, G. et al. Biological characteristics and metabolic phenotypes of different anastomosis groups of Rhizoctonia solani strains. BMC Microbiol 24, 217 (2024). https://doi.org/10.1186/s12866-024-03363-9

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