Morphology, pathogenicity, and molecular identication of Fusarium solani species complex (FSSC) associated with potato tubers

Potato (Solanum tuberosum L.) is among the top ve crops growing worldwide following cereals, rice, wheat, barley and corn due to its high carbohydrate content and adaptability. Potatoes are particularly valued in developing countries as a rich source of vitamins C and B6, starch and essential amino acids. Fusarium solani species complex (FSSC) is a common pathogen of potato, causing dry rot in the Upper Egypt. In this study were isolated and identied FSSC from potato tubers based on the morphological followed by molecular characteristics. 187 isolates of Fusarium solani were achieved from infected and non-infected potato tubers gathered from different markets in the Upper Egypt. Dependent upon the morphological characteristics, sequence data from amplifying β-tubulin and specic translation elongation factor (TEF-1α) genes, all of the selected FSSC isolates were divided into three major groups (F. keratoplasticum, F. falciforme and F. solani). All the tested FSSC were capable of producing amylases. All of the isolates were examined for their pathogenic ability on healthy potato tubers, which showed pathogenic effects; with lesion sizes were quite variable. F. solani SVUFs73 showed a highly virulent effect.

single spore of Fusarium colonies were inoculated rstly into petri dishes contained PDA medium, followed by inoculation of it in slants containing PDA medium and maintained at 4°C for further studies 22 . Colony morphology and microscopic examination were used for classical identi cation 23 .

Molecular identi cation of Fusarium isolates DNA extraction
Fusarium solani isolates were cultured in 250 ml asks containing 50 ml Potato Dextrose Broth (PDB) for 2-3 days using a rotary shaker for 25°C at 120-150 rpm. The mycelium was collected by ltration and ground to ne powder in liquid nitrogen. In an Eppendorf tube (1.5 ml) fty milligrams of the powder was put in it, then mixed with 0.7 ml 2 x CTAB buffer and vortex for 2min. Eppendorf tubes were incubated at 65 o C for 60-80 min, then 0.7 ml of chloroform was added and mixed brie y. After centrifugation at 15.000 rpm for 10 min, the supernatant was transmitted to a new tube mixed with 0.6 ml isopropanol and chilled to 20 o C, followed by another centrifugation step at15.000 rpm for 5 min at maximum speed. The supernatant was discarded and the resting pellet was washed twice with 1 ml of 70% ethanol, followed by another centrifugation for 3 min at maximum speed 15.000, after that dried and dissolved in 0.1 ml TE (10 mM Tris, 1 mM EDTA, pH 7.5) buffer (Moeller et al. 1992). The DNA quantity and quality checked by electrophoresis on a 1.4% agarose gel revealed with ethidium bromide and visualized by UV trans-illumination.

PCR ampli cation and sequencing
The primers used to amplify β-tubulin were Bt2a and Bt2b as described by Glass and Donaldson (1995). The primers used to amplify translation elongation factor-1α (TEF-1α) were EF1 and EF2 modi ed from 26 .
PCR reaction for both genes was carried out in PCR tubes containing 5 μL of the master mix (buffer, dNTP, Taq DNA polymerase, 2 mM MgCl 2 ) 1μL of the template DNA, 0.5μL of both forward and reverse primers and the volume was completed to 25 μL with PCR water. Ampli cation was performed in a thermal cycler (Flexigene, Techne, Cambridge, UK).
Polymerase chain reaction (PCR) cycles for β-tubulin were as follows: initial denaturation at 94 ºC for 1 min, 30 cycles of denaturation at 94 ºC for 30 s, annealing at 54 ºC for 30 s, extension at 72 ºC for 1 min and nal extension at 72 ºC for 5 min 25 . PCR cycles for TEF-1α, the cycle started with initial denaturation at 94 ºC for 5 min, 40 cycles of denaturation at 94 ºC for 1 min, annealing at 58 ºC for 1 min, extension at 72 ºC for 2 min and nal extension at 72 ºC for 10 min 27 . PCR product was observed in a 1.4% agarose gel, stained with ethidium bromide and visualized with UV transilluminator. Ampli ed products were puri ed, quanti ed and sequencing in Macrogen (South Korea).

Phylogenetic analysis
Sequences of β-tubulin and TEF-1α were edited by using chromas program and aligned using Clustal X included in MEGA version 6.0 28 . Phylogenetic analysis was conducted using combined dataset of β-tubulin and TEF-1α sequences. The Acremonium genus is closely related to Fusarium. Therefore, phylogenetic tree was rooted with Acremonium sclerotigenum (KC987128 and KT878381). The phylogenetic reconstruction was done using the neighbor joining (NJ) algorithm, with bootstrap values calculated from 1,000 replicate runs, using the software routines included in the MEGA software.
Screening of Fusarium solani isolates for amylase production Eighty eight isolates of Fusarium solani were screened for their abilities to produce extracellular α-amylase. Isolates were grown on solid starch yeast extract agar (SYE) medium with a composition (in g /L) of soluble starch, 5.0; Bacto-yeast extract, 2.0; KH 2 PO 4 , 1.0; MgSO 4 . 7 H 2 O, 0.5 and agar, 15 29 . Cultures were incubated at 28°C for 6 days. Using a sterile cork borer (10 mm diameter), the inoculums was obtained. For each fungal isolate, one sterile 100 ml Erlenmeyer ask containing 50 ml of the liquid SYE was prepared. Cultures were incubated at 28°C without shaking for 7 days after which the mycelium was harvested by ltration. Aliquots of 0.1 ml of a culture ltrate were pipetted into 10 mm cavities which were made in SYE plates. After 24 h incubation at 28°C, plates were ooded with iodine solution (KI, 15 g; I 2 , 3 g per liter of distilled water). A zone without blue indicates the production of amylase. In case of positive strains, the average diameter of clear zones (in mm) of the triplicates for each isolate was recorded.

Pathogenicity test of the selected isolates
The healthy potato tubers (Solanum tuberosum L.) were used in this experiment. Initially, tubers appearing healthy and similar in the size (100-120 g) were selected and washed to remove excess soil, surface sterilized in 50% sodium hypochlorite solution for 10 min and rinsed in 3 times of sterile distilled water (Lui and Kushalappa 2002;Lui et al. 2005) and then dried under laminar ow. Then the tubers wounded with a cork borer with a diameter of 5 mm to a depth of 5 mm 32,33 . An agar plug (5 mm diameter) containing active mycelium of Fusarium solani isolates extracted from the margin of a 3-day-old cultures grown on quarter of the quantity of PDA and placed into the wound, which was subsequently sealed with the excised plug of tuber tissue. Two tubers used for each fungal strain. All the wounded potato tubers were wrapped in black polyethylene bags 34,30 and incubated in the dark at 20°C for 3 weeks. As a control, tubers were inoculated with an agar plug only. Following incubation, tubers were cut longitudinally from the point of inoculation and the depth of internal necrosis was measured using electronic calipers. Re-isolations on PDA medium were attempted from all isolates. The depth of wound response in controls was also recorded for comparison. Tubers were cut through the inoculation points, and the degree of rot was estimated.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) using the Statistical Analysis System (SAS Institute, Inc., 1996). Means were separated by Duncan's multiple range test at P < 0.05 level.

Results
Morphological characterization of F. solani species complex 187 isolates of Fusarium solani were examined microscopically (Table 1). On PDA medium, aerial mycelia of all isolates were white at the initial stage, while the colonies became off-white, violet, purple and gray in the later stages. Single isolates of F. solani produced oval to kidney shape, microconidia measuring 9.7-23.4 x 2.9-5.6 μm. Macroconidia had 3-5 septa ranging 28.5-50.6 x 3.1-6.2 μm (Table 2& 3). On the same medium, chlamydospores were observed that appeared singly or in pairs and conidiophores were long monophialides.

The hierarchical analysis according to morphological characters of FSSC
The dendrogram was generated from 88 FSSC, the rst group (16 strains) and the second group (72 strains) according to morphological properties ( Figure 1).
From the hierarchical analysis, ( Figure 1) the isolates which had the same morphological properties were clustered together. The dendrogram divided into two clades.

Molecular characterization of Fusarium solani by ampli cation of β-tubulin and TEF-1α genes
To con rm the morphological identi cation of the studied isolates, molecular analyses were performed. β-tubulin was successfully ampli ed from 88 isolates of Fusarium solani species complex recovered from potato tubers samples. A single band of 350 bp was obtained by Bt2a and Bt2b primer pairs ( Figure 2). Translation elongation factor-1α (TEF-1α) was also successfully ampli ed from 83 Fusarium solani isolates and ve Fusarium solani isolates (SVUFf1, SVUFf2, SVUFs44, SVUFs86 and SVUFs90) have short sequence by TEF-1α gene, so didn't show in Phylogenetic analysis. A single band of 700 bp was obtained using EF1 and EF2 primer pairs ( Figure 3). All the sequences of β-tubulin and TEF-1α were deposited in the GenBank and their accession numbers were indicated in Figure (4) and (5). The β-tubulin and TEF-1α sequences of Fusarium solani isolates were subjected to GenBank database using BLAST search and the results were recorded as the most closely related sequences with high percentage of homology. TEF-1α showed the highly resolution comparable to β-tubulin in identi cation of Fusarium solani species complex Mega Blast analyses of the 2X consensus nucleotide sequence of β-tubulin and TEF-1α gene showed 99-100% similarity with several Fusarium species sequences of the same region deposited in GenBank. Neighbor joining tree using Mega 6 was used to study genetic relatedness of Fusarium strains (Figures 4 and 5).
β-tubulin dataset of F. solani species complex (88 F. solani) from the current study and 7 GenBank sequences ( Figure 4). The phylogenic tree ( Figure 4) revealed that, the strains of F. solani species complex were categorized into 3 clades. Phylogenetic analysis of this dataset resulted in the F. solani species complex clustered together in one cluster to the exclusion of out-group taxa.
SVUFs87 (F. solani) was found to be at a separate branch. The rst and second clade isolates were closer to each other than the remaining clades with strong support 92% bootstrap value. SVUFf1 and SVUFf2 (F. falciforme), SVUFs 24, 28, 34, 53, 57, 59, 71, 74, 75, 76, 77, 81, 85, 88 and SVUFs104 (F. solani) clustered together with Fusarium falciforme (KY776685 and KY776684) in the rst clade. Strains in this clade have the same morphological characters except isolates (SVUs53 and SVUs59), but all strains in this clade have morphological characters different from other Fusarium solani species complex in macroconidia, which was shorter than Fusarium solani species complex and these isolates gave the same results in the pathogenicity test (lesion sizes 10:16mm) and α-amylase production (level of this isolates less than ≤10 mm). The second clade comprised SVUFs102 of F. solani species complex. The third clade was divided into sub clades A, B, C and D. In the subclade A, SVUFs52 Fusarium solani was a base for all other Fusarium solani species complex presented in the third clade. Subclade B consisted from 2 strains (SVUFs62 and SVUFs72) of Fusarium solani species complex, which clustered together in one clade. Phylogenetic analysis was done by TEF-1α sequences for the 83 strains of F. solani species complex under study along with 5 GenBank sequences ( Figure 5). In addition to the out group sequence Acremonium sclerotigenum (KT878381). The phylogenetic tree revealed that, the tested strains could be categorized into six clades ( Figure 5).
Screening of Fusarium solani for α-amylase production 88 Fusarium solani isolates which were collected from potato tubers during this study were screened for production of αamylase qualitative assay depending on color change of Iodine indicator from blue to colorless in culture of Fusarium solani. All results recorded in Figure 6 and 7, each sample was tested in three replicates.
It was observed that all Fusarium strains produced α-amylase and production was more than ≥ 9 mm.

Pathogenicity of Fusarium solani
Fusarium solani were evaluated for their pathogenicity on healthy potato tubers. Measurements consisted into measuring the lesion's size in the pathogenicity test. Lesion sizes were completely variable and ranged from 0.0 mm to 35 mm ( Figure 6). Lesion size less than 10 mm wasn't pathogenic to potato tubers. The results of the pathogenicity test revealed that 88 isolates caused discoloration, necrosis and lesion of the tubers. Of the 88 isolates with putative dry rot symptom.
Tubers inoculated with F. solani SVUFs73 showed a mean lesion size of 32.7 mm, the highest average lesion size among all tested isolates Figure 6 and 8. Fusarium solani SVUFs73 showed higher pathogenicity effect than other isolates, based on average lesion sizes (Figure 8).
On the other hand, no differences were observed between the four isolates of F. solani species complex (SVUFs93, 96, 97 and 102), which showed a similar lesion size (22.7) mm ( Figure 8).

Discussion
This study is the comprehensive research for identi cation and genetic diversity of Fusarium solani , affecting the potato tubers in Upper Egypt. In this study, Fusarium solani were associated with potato tubers collected from the markets in Upper Egypt. This result in agreement with 3,14 showed that F. solani was the most frequently species associated with potato dry-rot in Egypt. According to other studies, Fusarium solani was the most frequent and aggressive among all Fusarium species isolated from potato tubers in different parts of the world 9 . This concurred with Chehri et al. 35 who assessed that F. solani was considered as in the virulent group. However, the genus of Fusarium solani is complex and morphological differences may be di cult to observe. Therefore, the DNA analysis is necessary for accurate identi cation and characterization of the species 36 .
β-tubulin sequences are often used to identify fungal species, and vast numbers of these sequences are already available in databases 37 . The results of Donaldson et al. 38 demonstrated the utility of β-tubulin sequences for phylogenetic studies in the genus Fusarium and a wide array of Ascomycetes. Sequence of translation elongation factor-1α (TEF-1α) gene always offered a ner resolution and separated strains of most Fusarium complex species at species rank 16 . For precise identi cation of FSSC in this study, a molecular systematic study of β-tubulin and TEF-1α genes was used. In this study, PCR assays with primers Bt2a/Bt2b and Ef1/Ef2 that amplify β-tubulin and TEF sequences enabled us to acquire the product with numerous species of F. solani. It was found that β-tubulin separated F. solani species complex under study to F. falciforme and F. solani. While, TEF-1α separated FSSC to F. falciforme, F. keratoplasticum and F. solani. Fusarium solani isolates were additionally clustered in the same main clade with several groupes which demonstrated intraspeci c variations. Fusarium solani also represent a species complex of 45 phylogenetic species which formed Fusarium solani species complex 39 . Inside F. solani species complex (FSSC), the isolates from soil and plant debris isolated worldwide are typically gathered in one or two phylogenetic species, known as FSSC3 and FSSC4 40,41 . Intraspeci c variations of F. solani have likewise been reported by Balmas et al. 42 in which two phylogenetic species, FSSC5 and FSSC9 were identi ed among 23 Fusarium solani species complex. These results came in agreement with Taha et al. 18 who performed β-tubulin gene sequencing of Fusarium isolates and phylogenetic analysis showed that the clade of F. solani divided into sub-groups. Mehl and Epstein 43 and Short et al. 44 reported that three of the most common species in F. solani species complex (F. keratoplasticum , F. falciforme and F. solani), showed a good deal of intraspeci c variation, and overlapping morphological traits. This result was in concurrence with Chehri et al. 45 who examined and phylogenetically analyzed 55 strains of the FSSC based on internal transcribed spacer (ITS) regions and partial translation elongation factor-1 (TEF-1α) sequences. They showed that the strains were characterized into four portrayed Fusarium species, to be speci c Fusarium keratoplasticum, F. falciforme, FSSC , and Fusarium cf. ensiforme. Also, the phylogenetic trees unmistakably distinguished rmly related species and particularly separated all morphological taxa. As indicated by Short et al. 44 , members from F. falciforme, F. keratoplasticum, and FSSC ordinarily were related with human infectious diseases while these strains were related with plants. These results were in agreement with Chehri et al. 35 who reported that based on the sequence data from translation elongation factor (TEF-1α) gene, all of the selected FSSC isolates were divided into two major groups. the rst group on molecular identi cation of FSSC strains isolated from potato tubers in Iran and the second group were Fusarium falciforme, which were reported for the rst time in Iran.
In this study, all the Fusarium solani were active in producing amylase especially F. F. solani (SVUFs73, 93, 96, 97, 102) (mean value ≥15 mm), whereas, low enzyme activity was exhibited by other isolates of F. solani (mean value ≤14mm). These results are in agreement with Kumar et al. 46 who reported that the primary and secondary screening results showed that a fungus isolated from degrading potato tuber, Fusarium solani, expressed maximum amylase production over other isolates. Subsequently, it was selected for amylase production and optimization studies indicated that as independent variable, pH-4 and incubation temperature of 30°C were found ideal for amylase production by F. solani.
All Fusarium solani isolates were pathogenic for potato tuber with different degrees. Isolate SVUFs73 of F. solani was highly pathogenic to potato tubers, whereas, other isolates which belong to the same species were weakly pathogenic to potato tubers. The mechanisms underlying this behaviour still vague. Some studies proposed that mycotoxins produced by Fusarium spp. play a key role in this regard 47,48,49 . These results were in agreement with the results published by 35 who con rmed that some isolates of F.solani were highly virulent and other isolates were virulent or nonvirulent. In addition, Ashour et al. 3 con rmed that, of the 10 isolates of F. solani with dry rot symptoms, one isolate was the most pathogenic while others were either less pathogenic (5 isolates) or non pathogenic, 4 isolates to potato tubers. According to Saber et al. 2 , 8 isolates of Fusarium solani were obtained from infected potato tubers of which 3 isolates were highly pathogenic to potato tubers, 2 isolates were avirulent, and other isolates were non pathogenic to potato tubers.

Conclusions
Based on morphological, microscopic characteristic and molecular identi cation by sequencing of β-tubulin and TEF-1α genes, we proved the presence of Fusarium keratoplasticum, F. falciforme and F. solani on potato tubers in Upper Egypt. Based on our knowledge and research, this is the rst comprehensive report on identity (morphological and molecular), pathogenicity and distribution of members of F. solani species complex from potato tubers in Upper Egypt. TEF-1α gene in molecular identi cation of FSSC was better than β-tubulin. FSSC demonstrated the most aggressive properties and amylase production, with present strong relationships between pathogenicity and α-amylase enzyme production.    Neighbor-joining phylogenic tree of 83 isolates of Fusarium solani species complex resulting from the sequence results of TEF-1α gene using Acremonium sclerotigenum as out group.