1.
Introduction
Rhizoctonia solani is a soil-borne fungus that belongs to the phylum Basidiomycota [1,2]. This fungus severely impairs the quality and quantity of potato production, resulting in significant economic losses [3,4]. However, a variety of mild symptoms, such as misshaped tubers, growth cracks, netted scab, dry core, and tuber greening, which manifest more frequently than any other symptoms, contribute to yield losses [5,6]. Observed moderate symptoms include sunken lesions on stems (cankers), necrotic lesions on stolons and roots, as well as tuber sclerotia (irregularly shaped masses of fungal mycelium also known as black scurf), which cause seedling mortality and reduced tuber output. In severe infections, the fungus can girdle and kill the stems, damaging the entire plant [1,7]. Other fungi, such as Fusarium spp. and Alternaria spp. also cause problems in potato crops and inflict various damages but to a lesser extent than Rhizoctonia spp. which is the more prominent potato pathogen [8,9]. Tuber quality is often reduced by the development of sclerotia on progeny tubers, and thus, the product's marketability is adversely affected, particularly for pre-packaged potatoes [5]. In some cases, marketable yield losses approaching 30% have been reported [10]. The potato (Solanum tuberosum L.) is one of the most important non-grain food crops in the world [11]. In Jordan, potatoes are widely grown in a wide variety of environments. The potato crop, as with many other agricultural and horticultural crops, is subjected to attacks by species of Rhizoctonia, a globally ubiquitous fungus genus [1,3]. Due to its moderate climate, the Jordan Valley is a major producer of potatoes in the autumn and winter. In contrast, the rain-fed regions (the Highlands) are mainly cultivated in the spring. To cultivate their crops, farmers use a variety of imported potato tubers most of them came from the Netherlands, France, and Germany without assurances that these tubers are free of R. solani. According to Abu El Samen and Al Bodor [12], R. solani was present in 42.5% of the tested 109 samples of imported potato tubers from Europe.
Rhizoctonia solani is a complex species composed of 13 anastomosis groups (AG1-AG13, with AG B1 being a subset of AG-2 B1) [13]. Traditionally, the AG grouping is assigned phenotypically based on hyphal interactions, where hyphae of isolates belonging to the same AG are able to anastomose [1]. However, such a traditional method does not offer reliable results, particularly for designating group subsets [3]. Other parameters, including pathogenicity, biochemical traits, and modern genetic markers, have been used to more reliably and definitively assign the AG groupings [14]. Molecular approaches for accurately identifying Rhizoctonia subgroups have been increasingly used to assign each isolate to its AG group. Polymerase chain reaction (PCR)-based techniques for amplifying the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) are frequently used in conjunction with phylogenetic analysis to understand the genetic relationship among isolates [3,15]. This method is thought to be superior in terms of assigning anastomosis AG grouping to hyphal interactions. There is relatively little data given about the biology of the disease and the relative importance of the various AGs in the etiology of Rhizoctonia diseases in potatoes in Jordan as well as other countries. Knowing this information is crucial for assessing the use of particular fungicides that may work effectively against specific AGs. Furthermore, collecting such information will help predict the disease's severity once it appears and the correct approach to diagnose and treat it [14]. According to studies, different AGs have different hosts. Hence, they are designated by an abbreviation. For example, the AGs for potato (AG3-TP), tobacco (AG3-TB), and tomato are (AG3-TM). Each is consequently associated with various disease symptoms.
There are no projections of the impact of Rhizoctonia diseases on tuber quality and yield in Jordan, and farmers are frequently unaware of the underlying causes of their potato crop damage. The objective of this study therefore, was to isolate and identify all causative agents affecting the potato crops in the Jordan Valley using molecular techniques and to study the pathogenicity of specific R. solani AGs to potato and other vegetable plants.
2.
Materials and methods
2.1. Sample collection and fungal isolation
One hundred fifty potato plants and tubers with typical R. solani symptoms (black scurf and canker on stems, stolons, and roots) were obtained during March and April of 2017 from 15 transects within fields from three geographically different locations in the Jordan Valley. These locations represent the most important potato-growing areas in the Jordan Valley (Table 1).
The collected samples were brought to the plant pathology laboratory in the faculty of agriculture at the Jordan University of Science and Technology to isolate the pathogen and examine its phenotypic and pathogenic traits. Infected plant materials with obvious disease symptoms were rinsed in running water and dried for four hours in a laminar flow cabinet in order to separate Rhizoctonia and other pathogens. Small pieces of infected tissue, 4 mm in diameter and 5 mm deep, were excised using a sterile scalpel blade and plated onto petri plates containing 1.5% water agar (WA) supplemented with streptomycin sulfate (Sigma-Aldrich) at 50 mg/liter. The plates were incubated at 25 ℃ for 48 hours. The colonies of each isolate that was determined to be a Rhizoctonia spp. were examined under a microscope, and fungal hyphal tips were collected. Following the procedures outlined by Carling and Leiner [16], tissue samples were placed onto potato dextrose agar (PDA; Biolab, Hungary), and were incubated for three days at 22 ℃.
2.2. Molecular identification
DNA was extracted from 57 pure fungal samples that were phenotypically identified as Rhizoctonia spp. according to the method described by Liu et al. [17]. The quantity and quality of DNA samples were detected using NanoDrop (Thermo Fisher scientific cat ID: ND-2000) and gel electrophoresis, respectively. Working DNA solutions of 20 ng/µL were prepared, and both the stock and working solution were stored at −20 ℃.
The ITS region for each isolate was amplified using the universal primers; ITS1-F (5'-CTTGGTCATTTAGAGGAAGTAA-3') [18] and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3') [19] that anneal to the flanking 18S and 28S rDNA genes. PCR reactions were conducted under the following conditions; 95 ℃ initial denaturation for 5 minutes, 35 cycles of 1 min 95 ℃ denaturation, 30 sec 45 ℃ annealing, 1 min 72 ℃ extension, 72 ℃ final extension, and holding at 4 ℃. Each primer (1 µL) was added to the reaction from a 10 µM working solution alongside with 2xPCR master mix solution (Ⅰ-MAX Ⅱ) (cat ID 25266), 20 ng template DNA, and an appropriate amount of nuclease free water to reach 20 µL final volume. A negative control sample was used containing all the components except the DNA template was used to check for any contamination.
PCR amplifications were analyzed using agarose gel electrophoresis stained with ethidium bromide. Prior to sequencing, PCR products were cut from the agarose gels and purified using Zymoclean Gel DNA Recovery Kits (cat IDD4007).
Sequencing reactions were carried out using BigDye™ Terminator v3.1 Cycle Sequencing Kit (cat ID 4337455) and analyzed using the genetic analyzer 3100 DNA sequencer located in the Princess Haya Biotechnology Center, Jordan University of Science and Technology (JUST).
NCBI BLASTn database was used to check the identity of each sequence and to find the closest match based on maximal percentage identity. Confirmed sequences were deposited in GenBank and were assigned accession numbers.
2.3. Phylogenetic analysis
Two phylogenetic trees were constructed, the first of which (Figure 1) displayed the phylogenetic relatedness between all 57 isolates, while the second of which (Figure 2) displayed only the 21 Rhizoctonia isolates (Figure 2). Both trees were constructed with 5 Rhizoctonia sequences representing out groups: an out group belonging to a different AG group (AB054845.1, (AG-2-1), an AG3 group infecting tobacco (AB000004.1 AG-3-TB), an AG3 group infecting tomato (AB000023.1(AG-3-TM), and two isolates of an AG3 group infecting potatoes isolated from other parts of the world (KX631235.1) AG-3-PT, and KX631236.1 (AG-3-PT) [20].
Phylogenetic trees were constructed using the Neighbor-Joining method [21]. The optimal tree with a sum of branch lengths of 0.22 mm is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [22]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura 3-parameter method [23] and are in units of the number of base substitutions per site. The phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis [MEGA X] software [24].
2.4. Determination of somatic compatibility grouping
All the confirmed Rhizoctonia isolates as AG-3PT by the ITS rDNA sequencing were used in this study. Slides with a thin covering of water agar used to detect microscopic somatic compatibility reactions (WA Oxoid) were made for the pairings. The interaction was evaluated under a light microscope using safranin O staining [25]. According to the method of Carling [26], we determined the number of fields in each interaction category (C0, C1, C2, and C3) in 15 visual fields. The fusion frequency has been determined as %FF = (A*100)/B. According to MacNish et al. [27], the macroscopic somatic reactions were defined as merge and tuft according to predefined categories after the plates were incubated at 15 ℃ for 21 days and assayed.
2.5. Pathogenicity tests
Twelve plant species were tested as potential hosts for three isolates confirmed as AG-3PT, which were selected randomly from each of the three regions to be tested for their pathogenicity. These were seedlings of carrot (Daucus carota L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), soybean (Glycine max), corn (Zea mays L.), Wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.), okra (Abelmoschus esculentus), cauliflower (Brassica oleracea L.) and potatoes (Solanum tuberosum L.). The seeds were germinated in peat. The seedlings were planted in pots containing sterile sand. After five days, each plant was inoculated with a piece of a Rhizoctonia growth with a diameter of 5 mm from the fungal colonies' margins. This part of the mycelium was placed one cm deep next to the seedling's stem and covered with sterile quartz sand to prevent drying. As per Carling and Leiner [28], potato plants were used as a positive control. Plants were incubated at 20 ℃ for a 12 hour photoperiod in a growth chamber, irrigated with distilled water every 2–3 days, and fertilized with a nutrient solution weekly [29]. The symptoms on the stems and roots were evaluated after 15 days of inoculation. On the stems, the symptoms were classified as small superficial lesions (SS L = lesions size < 5 mm) or cankers (C). On the roots, disease incidence, and lesion categories were characterized as on the stems. Re-isolation was carried out on infected and healthy plant tissues to confirm the presence of fungi in the tested plants.
2.6. Statistical analysis
The experimental design for the pathogenicity test was a complete randomized block design with three replications. The data were subjected to an analysis of variance, and the treatment means were separated by Duncan's multiple range test (α = 0.05). The data were analyzed using GENSTAT 14th Edition (VSN International).
3.
Results
3.1. Identification of Potato infecting species by ribosomal DNA sequencing
For primary identification, morphological characterization was performed on all 57 isolates. Phenotypically, all conformed to Rhizoctonia. However, their identity was determined by sequencing the internal transcribed spacer (ITS-rDNA). Surprisingly, only 21 isolates were confirmed as R. solani, while the rest were distributed among different fungi species described in Table 2. It was noticed that Aspergillus spp. and Fusarium spp. were the most predominant after Rhizoctonia spp.
The obtained rDNA sequences were deposited in the GenBank, and accession numbers were assigned (Table 3). All the Rhizoctonia isolates (100%) were of anastomosis group 3 (AG-3PT). Among these, 12 isolates were isolated from stem cankers, while the other nine isolates were isolated from sclerotia on the mother tubers.
3.2. Phylogenetic analysis
The phylogenetic tree was constructed for all 57 isolates (Figure 1) to study the clonal relationship among all the fungi that grow and cause diseases in potatoes. As expected, all strains of R. solani clustered together along with the out group isolates that infect potatoes. Indeed, the phylogenetic tree separated each species into a single cluster. Except for a few isolates that appeared to cluster with isolates from different species, this was true for the majority of fungal species. The other tree was constructed for the 21 Rhizoctonia isolates (Figure 2) and five other isolates obtained from published sequences. The bootstrap values on all nodes showed no significant differences between our isolates, which reflect their common source with the two AG3 isolates from China [30] that appeared within the Jordan AG3 isolates. However, the other three outer isolates, the AG3 isolates (TB and TM) and the out group AG-2-1 isolates, appeared distant from the potato AG3 group isolated in this study.
3.3. Hyphal interactions
The hyphal fusion ratio of 15 isolates belonging to AG3 ranged from 45 to 96%. In addition, a high percentage of isolates have frequencies between 47 and 74%, which indicates a lack of harmony between individuals indicating that the anastomosis reaction was positive and the frequency of hyphal fusion exceeded 50%. About 86.7% of isolates pairs were evaluated, and the formation of tufts was common in the macroscopic somatic assay indicating a somatic incompatibility between the isolates. However, the perfect fusion occurred in only 13.3% of the pairs tested, indicating their compatibility (Figure 3). Table 4 presents the results of the hyphal interaction.
3.4. Pathogenicity tests
Isolates of R. solani AG3-PT caused large lesions (>10 mm) on the stems of potato, tomato, pepper, and okra, while their roots did not show any symptoms. As for the roots of pea, bean, soybean, wheat, barley, corn, and carrot plants, the AG3-PT isolates caused numerous small lesions. Furthermore, the AG3-PT isolates caused lesions on the stems of soybean, barley, corn, and carrot plants while not causing any lesions on pea, bean, or wheat. The cauliflower showed no signs of disease (Table 5 and Figure 4).
4.
Discussion
This is the first study in Jordan to describe and characterize the disease-causing pathogens of potato crops and the identification of these pathogens both at the phenotypic and genotypic levels. Despite the fact that R. solani is generally thought to be the major pathogen infecting potatoes, several other fungi species were also isolated from infected potatoes grown in the Jordan Valley. The types of these pathogenic fungi and their distribution are presented in Table 2. Many of these fungi are known plant pathogens and could inflect various degrees of damage to potato plants as well as to other crop plants [31]. The most prevalent species among these isolated pathogens was R. solani (35%). All of the isolates' species identities were determined by ITS rDNA sequencing (Table 2). As inferred from the ITS rDNA sequences, R. solani isolates found in Jordan that were recovered from various potato diseases were all AG3-PT (100%). This result confirms previous reports about the predominance of AG3-PT on potato crops [32,33,34,35,36,37]. Furthermore, the phylogenetic analysis revealed that the isolates of R. solani are associated with each other with a low degree of heterogeneity which could be due to the differences in the locations of samples collection (north, central, and south of Jordan Valley). Interestingly, AG3-PT isolates from China clustered with the AG3-PT isolates from Jordan. In contrast, the cluster analysis showed that AG3-TB and AG3-TM did not cluster with the AG3-PT isolates, indicating the low heterogeneity among the AG3-PT and the vast difference between the AG3-PT and the AG3-TB or AG3-TM as inferred from the phylogenetic analysis. In addition to its high virulence on potatoes, one of the reasons AG3-PT is so common in potatoes is its ability to form sclerotia (black scurf) on tubers, inflicting severe damage to the corp. The results are in accordance with the observations of Lehtonen et al. [38] who postulated that AG3-PT is much more efficient in producing sclerotia on tubers than other AG subgroups, reflecting the highly specialized nature of AG3-PT in infecting potatoes and thus being more aggressive. Nonetheless, other AG groups inflict severe damage to other plants, such as tomatoes and tobacco [39,40].
The correlation between microscopic killing anastomosis (C2 reaction) and perfect fusion (C3 reaction) with the corresponding macroscopic somatic interactions "tuft" and "merge" has been reported for some R. solani groups, such as AG8 [27] and AG3-TB from tobacco [41], as well as R. solani AG3-PT from potatoes [3]. Nevertheless, the macroscopic somatic interactions were not good predictors for the microscopic anastomosis reactions between isolates of the soybean-infecting pathogen R. solani AG1-IA [42]. Similar to previous reports on the soybean-infecting pathogen, perfect fusion and killing anastomosis reactions at the microscopic level were not distinguished in this study. Thus, a correlation between macroscopic and microscopic for R solani AG3-PT was not established. The results of this study indicated incompatibility between the tested isolates, as some fusions appeared in large quantities of aerial mycelia (tuft) (Fig 3a, b) indicating a high level of genetic diversity among the isolates (Figure 3a, b). Somatic compatibility existed in only 13.3% of the pairs. Further, the obtained results indicated that R. solani AG3 is capable of infecting different plant species and inflicting severe damage, which is consistent with previous reports that even specialized mold pathogens have shown some levels of damage in hosts distinct from their own original host [3,16,39,43,44,45,46]. Indeed, these results have shown the need to adopt crop rotation strategy and the use of specific fungicides to control R. solani from accumulating in the soil and infecting other plants that showed readiness to be infected by this pathogen. In addition, even though the fungal inoculums could remain viable on plant debris and hidden roots for a long time, planting cauliflower as a non-host to interrupt the pathogen's life cycle can be a part of the fighting strategy against this mold [31].
This work has provided knowledge about the R. solani groups in different locations in Jordan. Further, it has confirmed the predominance of AG3-PT associated with potato disease in Jordan. The significance of determining the AG group for a location could be used as a guide for selecting an effective disease management regimen. This belief stems from the idea that genetic differences exist among the different AG groups, which might be reflected in susceptibility/resistance to a certain fungicide [34,40]. Further, understanding the host range for the different AG groups is of prime importance for fighting strategies [44].
5.
Conclusions
The internal transcribed spacer (ITS) sequencing and the phylogenetic analysis of the Rhizoctonia solani isolates confirmed the identity of the isolates in which AG-PT3 was the major pathogen affecting the potato crop in Jordan.
The obtained results for the different AG groups can be used to implement strategies for combating this fungus. Crop rotation is one of the regimens that can break the fungi's life cycle by growing non-host-specific crops and choosing the most appropriate fungicide for a particular AG group.
Acknowledgments
The authors would like to acknowledge Jordan University of Science and Technology for providing facilities to complete this project in its laboratories.
Conflict of interest
The authors declare that they have no conflict of interest.
DownLoad:
