Vegetative Growth and Molecular Identification of Fusarium equiseti Isolated from Wilt Disease of Centella asiatica L. in Bangladesh

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30 ̊C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.


Introduction
Centella asiatica (L.) is a prostrate, faintly aromatic, stoloniferous, perennial, creeper herb, commonly known as Thankuni in Bengali [1]. It is found almost all over the world and flourishes extensively in shady, marshy, damp and wet places such as paddy fields, river banks forming a dense green carpet. The leaves, which are edible, are in yellowish-green color, thin, alternate with long petioles, and quite characteristic reniform, orbicular, or oblong-elliptic shapes with seven veins. The plant grows horizontally through its green to red stolones which combine with each other and roots underground [2].
It is an important plant with wide range of traditional, medicinal and therapeutic values and is utilized as a source of food, beverages, medicine. This is a key element of numerous compound formulations used for curing gastrointestinal disorders, cutaneous problems and for revitalizing brain cells. In Southeast Asia, it is traditionally used for the treatment of a wide variety of disorders such as skin diseases, rheumatism, inflammation, syphilis, mental illness, epilepsy, hysteria, dehydration, and diarrhea [1] [3]. The chemical constituents of C. asiatica have a very important role in medicinal and nutraceutical applications and it is believed due to its biologically active components of triterpenes saponins. The triterpenes of Centella are composed of many compounds including asiatic acid, madecassic acid, asiaticosside, madecassoside, brahmoside, Brahmic acid, brahminoside, thankiniside, isothankunisode, centelloside, madasiatic acid, centic acid, and cenellicacid. Due to their importance, they have been used as the biomarker components for quality assessment of Centella [4]. According to Zainol et al. [5] the highest concentration of phytochemicals was found in the leaves relative to the petioles and the roots. It is also rich in vitamin C, vitamin B1, vitamin B2, vitamin A and niacin carotene.
The demand for C. asiatica in different cases especially for medicinal purpose is increasing day by day. Nowadays, it is cultivated in different parts of the world. But the major problem in its cultivation is different diseases caused by microorganisms, especially by fungi. The diversity of the endophytic fungi was classified as a medium diversity. The highest diversity of the endophytic fungi was obtained from the roots, followed by the leaf, stolon, and petiole. Ceratobasidium sp., Colletotrichum sp., C. destructivum, Fusarium solani, Alternaria and Curvularia, were the most dominant endophytic fungi in plant organs of C. asiatica in Malaysia. Souza and Santos [6] reported that Colletotrichum spp. and Fusarium spp. were the most common endophyte fungi that cause major diseases in various medicinal plants. Fusarium sp. is associated with wilt disease of C. asiatica which is characterized by browning of the leaf and dark brown lesion development on leaf surface finally the leaf become dry out.
Assessment of genotypic and phenotypic diversity is helpful to distinguish of pathogenic fungi. Different genetic tools have been introduced such as ITS sequence, restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) and small subunit rhibosomal DNA (rDNA).

Objectives
The objectives of this study was undertaken deal with the accurate identification of wilt disease caused by Fusarium equiseti associated with Centella asiatica based on morphological, growth characteristics and molecular approach.

Sample Collection
Wilt disease of Centella asiatica was collected from different areas of Jahangir-

Fungus Isolation and Morphological Characterization
Fungus was isolated through tissue planting method. Infected parts of C. asiatica were cut in to small piece about 0.5 cm in length in such a manner so as to include both fungal infected and non-infected tissues in piece. Then sterilization was done using NaOCl (5%) solution for 5 minutes and rinsed with distilled water several times. Four pieces of samples were placed into PDA medium and were incubated under 12/12 hours dark and light condition at 25˚C ± 2˚C for 10 days. Mycelial growth of growing fungus colony was transferred to fresh PDA plates as well as PDA slants to obtain a pure culture. The pure culture of the isolated fungus was identified microscopically using standard methods based on colony morphology, mycelium and conidia [10]. were used to assay the mycelial growth of the pathogen according to Sultana et al. [11]. The media will be adjusted to pH 6.5 before autoclave. Different temperatures (15˚C, 20˚C, 25˚C, 30˚C and 35˚C) were maintained for the mycelial P. Akter et al.

Effect of Culture Media, Temperature and pH
growth of the pathogen on PDA in an incubator. The mycelial growth was recorded at 7 days post inoculation (dpi) respectively [8]. Different pH levels viz., 5.0, 6.0, 7.0, 8.0 and 9.0 were used [9]. The medium was adjusted to pH 4, 5, 6, 7 and 8 with the addition of 1 N NaOH or HCl before autoclave and it will be incubated at 30˚C for 7 days. Radial growth of mycelia on each Petri dish was measured at 3 directions according to Alam et al. [8].

Molecular Characterization
After microscopic examination, molecular characterization was done with the help

Morphological Characteristics of Fusarium equiseti
Aerial mycelia were abundant and pinkish-white (Figure 1(a)). Pale to violet pigmentation formed in agar and the colony centers often were heavily pigmented. Sporodochia were absent. Microconidia were abundant in false heads or oval, obovoid with a truncate base, elliptical in shape (Figure 1(b)). Macroconidia were extremely sparse and difficult to find but, where present, they were typically three-septate, fusiform with tapered apical cell and with poorly developed foot-shaped basal cells.

Effect of Culture Media, Temperature and pH
The effect of culture media viz., SGA, YEA, HPA, MA, CA, PDA, HA and PSA on mycelial growth of Fusarium equiseti have been presented in Figure 2 and Figure 3. The results showed that the highest mycelial growth (89.25 mm) of F. equiseti was recorded on PDA medium which was followed by CA medium and the lowest growth (40.25 mm) was measured in HA medium.
PDA is one of the most commonly used culture media because of its simple formulation and its ability to support mycelial growth of a wide range of fungi.
Several workers stated that PDA to be the best medium for mycelial growth of   fungi [13] [14]. The present studies confirm the reports of earlier workers doing their physiological studies related to suitable media for growth and sporulation of Fusarium sp. Khan et al. [15] reported  Figure 4. The data informed that the highest growth of F. equiseti was recorded at 30˚C, followed by 35˚C. In our experiment, F. equiseti grew the maximum at 30˚C which is consistent with the previous findings of Iwen et al. [18] who cited that the highest mycelia growth and sporulation of Mucor circinelloides registered at 30˚C, a sudden fall in mycelial growth and sporulation observed at 30˚C and 35˚C. M. circinelloides grew with well-developed mycelium at 25˚C to 35˚C. This showed that temperature has no effect on the growth of the mycelial growth of F. equiseti. There is always an optimum temperature for the best growth of any fungus as temperature has a regulatory effect on fungal growth and development. Daami-Remadi et al. [19] observed that thermal optimum of 30˚C was suitable for maximum mycelial growth of Fusarium solani. Singh et al. [20] noticed that the optimum temperature for growth and sporulation of Fusarium solani was 30˚C. American Journal of Plant Sciences pH is also one of key criteria for understanding the ecology of spoilage fungi, however, in present study, the experimental plates were incubated at five different pH level viz., 5, 6, 7, 8 and 9. Maximum mycelia growth (82.25 mm) of Fusarium equiseti was recorded at pH 7 and followed by pH 8 and pH 6, while minimum mycelial growth was found at pH 8 and pH 9, respectively ( Figure 5).
Sharma et al. [21] studied the effect of pH on the growth of F. oxysporum and reported that Fusarium sp. could sporulate grew well at 5.5 pH. Gangadhara et al. [22] reported that the maximum growth of Fusarium oxysporum occurred at pH 5 and pH 6 in vitro. Similar results are also reported the earlier who obtained the maximum growth of Ceratocystis fimbriata at pH 7.5, followed by pH 7.0 and pH 8.0 [23]. Yadahalli et al. [24] noticed the maximum mycelial growth of Ceratocystis paradoxa when the pH of the medium was between 6.0 and 7.5.
Result indicated that this fungus grew well at neutral condition.

Molecular Characterization
The internal transcribed spacer regions of fungal rDNA are highly variable sequences of great importance in distinguishing fungal species by PCR analysis.  spacer of rDNA is considered as a variable region among the species and even among the strains [8].
The ITS region was amplified using ITS4 and ITS5 primers and sequenced.
Phylogenetic tree based on the nucleotide sequences of the ITS regions in thirty six fungal taxa were selected from the NCBI database for phylogenetic analysis.
In maximum parsimony tree there are seven different clusters were found in the phylogenetic tree. Per cent homology of rDNA sequence of ITS region  1, JUF0043). The results indicated that all the individual species of Fusarium equiseti belong to major cluster. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. The size variation was caused by differences in the number of nucleotides, revealing that these strains are clearly distinguishable from each other based on the ecological distribution, substitution, and insertion or deletion polymorphisms of the base position [8]. Alam et al. [7] reported that ITS sequences are genetically constant or show little variation within the species, but vary between species in a genus. The genetic diversity detected  within groups is probably due to an efficient gene flow and to a high genetic compatibility within the strains tested. Based on the molecular evidence, it is clearly indicated that our studied fungus is Fusarium equiseti.

Conclusion
Fusarium equiseti was identified as a causal agent of wilt disease in Centella asiatica based on classical fungal taxonomy and molecular characterization. PDA was the best culture medium for the mycelial growth and development of F. equiseti. Temperature 30˚C was optimum, tested fungi grew well in the neutral condition. The PCR product of the ITS region of F. equiseti was 535 bp. Sequence analysis showed that 5.8S of rDNA sequences were identical. The reciprocal homologies of the ITS region sequences ranged from 98% to 100%.