1. Introduction
The cultivation of medicinal plants has become one of the vital earning sources for rural people in Bangladesh. Aloe vera plants are being cultivated in the northern parts of Bangladesh. These plants are used in medicine preparation, cosmetics industries and juice [1]. The contamination in leaves with fungi and other microbes is of public importance. Besides, fungal pathogens produce mycotoxins, which is a human health concern. Due to the succulent nature of the A. vera plants, they are susceptible to various pathogenic microorganisms responsible for qualitative and quantitative loss and incur the lower production of the plant. Some fungal diseases of Aloe have been reported worldwide and in Bangladesh. For instance, leaf spot disease caused by Alternariaalternata [2]; Nigrosporaoryzae [3]; Fusariumoxysporum [4]; Colletotrichumsiamense [5]. Generally, mycologists use classical taxonomy to identify fungi. However, diagnosing the fungi at species rank accurately is challenging due to fewer distinct taxonomic features. Now, mycologists use molecular data to identify fungi using sequencing of nuclear ribosomal DNA, translation elongation factor, beta-tubulin gene, etc. [6]. Three species—Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae causing grey blight disease of tea in China was described using molecular techniques as mentioned above. The ITS is the most useful marker for detecting fungi at species rank due to the fastest evolving portion of the rRNA cistron. Besides, internal transcribed spacer region has been chosen as the official barcode to identify fungi due to their difference between intraspecific and interspecific variation [7] .
The culture media plays a vital role in the growth, development and sporulation of fungi. Besides, relevant environmental factors such as temperature, light, etc. are essential for the germination of fungal spores, the development of germ tubes, thereby disease development. Farmers generally use fungicides to manage fungal diseases. However, harmful chemical fungicides are detrimental to the environment, animal and human health. The use of biocontrol agents could be an effective alternative to chemical pesticides. Therefore, an experiment was conducted to detect the causal agent of A. vera leaf spot disease; to study the influence of fungal culture media, temperature, light on the isolated fungus; to evaluate the efficacy of selected bio-control fungi and chemical fungicides against the isolated fungus.
2. Materials and Methods
2.1. Isolation and Identification of the Fungus
Diseased A. vera leaf samples with diagnostic symptoms were collected from the Northern part (Natore district) of Bangladesh and the Botanical Garden, Jahangirnagar University, Bangladesh. Tissue planting method was employed to isolate fungus and was identified based on colony morphology and morphological features of mycelia and conidia [8]. The pathogenicity test of the isolated fungus was carried out following the modified “detached leaf technique” [9].
Two universal primers ITS4 (5-TCCTCCGCTTATTGATATGC-3) and ITS5 (5-GGAAGTAAAAGTCGTAACAAGG-3) were employed to amplify the fungus’s ITS region. [10]. The PCR reaction was performed according to Sikder et al. [11]. The purified PCR products were sequenced in the Sanger sequencing platform, First BASE Laboratories, Sdn Bhd, Malaysia. Sequencing data were submitted to NCBI and received an accession number. Sequence data were compared with other nucleotide sequences after retrieving from the NCBI GenBank database. The maximum likelihood tree was generated using MEGA 6 software.
2.2. Effect of Culture Media, Temperature and Light on the Vegetative Growth of the Fungus
To assess the fungal mycelial growth pattern, six discrete fungal culture media, namely Potato Dextrose Agar (PDA), Potato Sucrose Agar (PSA), Carrot Agar (CA), Richard Agar (RA), Honey Peptone Agar (HPA), Honey Agar (HA) were used [12]. The five different temperatures conditions were tested to evaluate the mycelial growth of the fungus was according to Sultana et al. [13]. The influence of three photoperiodic conditions on the vegetative growth of the fungus was also investigated [14]. Data were recorded at 7 days post-incubation (dpi).
2.3. In Vitro Mycelial Growth Inhibition of the Fungal Pathogen
The vegetative growth inhibition of the target fungus was assessed using the dual culture technique, in which three biological control agents namely—Trichoderma reesei, Trichoderma harzianum, and Trichoderma asperellum were used [15]. The efficacy of three different synthetic fungicides Tilt 250EC @ 100 ppm, 250 ppm and 500 ppm; and Ridomil gold MZ 68 WP and Amistar Top 325 SC @ 250 ppm, 500 ppm, and 750 ppm were evaluated on the isolated fungus by poison food techniques under laboratory conditions [16]. The PDA plate containing the tested fungus without any bio-control agent or fungicide was served as control. The percent vegetative growth inhibition of the fungus was estimated at 7 dpi [17].
2.4. Statistical Analysis
Data on the effects of fungal culture media, temperature, light, bio-control agents, and chemical fungicides on the mycelial growth of the fungus were checked for normality and homogeneity of variance. Data were analyzed using one-way ANOVA with Duncan’s Post-Hoc test in SPSS-16.
3. Results and Discussion
3.1. Isolation and Identification of the Fungus
Pseudopestalotiopsistheae (P. theae) formed a rounded spot with black color in the center and a brown margin on the plant’s upper leaves (Figure 1(A)). The fungus produced white color colony with an irregular margin and formed sclerotia on the PDA medium (Figure 1(B)). Acervuli were scattered, globose to lenticular. Conidia had three median cells with pigmented light brown and dark
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Figure 1. (A) Symptoms of P. theae causing leaf spot disease in the leaf of Aloe vera plant; (B) Vegetative growth of the fungus on PDA medium; (C) Microscopic view of conidia (100×); profiles of amplification of rDNA-internal transcribed spacer region of the targeted fungus obtained with ITS4 and ITS5 primers.
brown margin. The apical cell of conidia contains 3 flagella and the basal cell contains single flagella. Conidia are fusiform in shape (Figure 1(C)). Similar morphological features were also described by another researcher [8]. The pathogenicity test confirmed the isolated pathogenic fungus—P. theae through Koch postulates. The typical dark brown and black irregular spots and lesions were observed on the artificially inoculated leaves after 7 - 10 days of inoculation.
The PCR product of the studied fungus was approximately 650 bp in size (Figure 1(D)) which was further confirmed via Sanger sequencing. In BLAST search, our studied organism MH333081.1 Pseudopestalotiopsistheae showed 98% sequence similarity with previously identified species, KM111476.1 Pseudopestalotiopsistheae; KX757714.1 Pseudopestalotiopsiscamelliae-sinensis; KX757707.1 Pseudopestalotiopsiscamelliae-sinensis; HQ832793.1 Pseudopestalotiopsistheae and MF495464.1 Pseudopestalotiopsistheae. The clustering of the species confirmed our organism as Pseudopestalotiopsistheae There were three major clades in the maximum likelihood (ML) tree; the first clade consists of both Pseudopestalotiopsiscamelliae-sinensis andPseudopestalotiopsis theae i.e., theae group in which our studied organism located with highly similar taxa (Figure 2). The second clade had a group of species belonging to the genus-Neopestalotiopsis. The third clade had different species of the genus-Pestalotiopsis (Figure 2).
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Figure 2. A maximum-likelihood tree of the 18S rDNA sequences of the studied fungal organism with retrieved NCBI sequence data. Our studied organism (MH333081.1) has been marked as “This study”.
3.2. Growth Characteristics of the Studied Fungus
The vegetative growth pattern of the fungus on different solid culture media viz., PDA, CA, RA, PSA, HPA, and HA was distinct and there were significant differences among the fungal culture media (Figure 3(A)). In our investigation, RA and PSA gave the maximum mycelial growth of P. theae compared to commonly used PDA media. Although RA medium supported the highest mycelial development, sclerotium formation was the maximum on PSA medium. Our results agree with the findings of previous worker, who cited the radial mycelial growth rates of Pestalotiopsis spp. (Pestalotiopsis fici, P. guepinii and P. palmarum) were affected by culture media [18]. The PSA was the most favorable for fast radial growth of mycelium than Pestacia Leaf Agar and Water Agar media. The V8 juice agar supported the most rapid mycelial growth rate of Pestalotiopsismicrospora compared to PDA and malt extract agar [19] . In a lab bioassay, Pestalotiopsisfunerea has been reported the most remarkable growth rate of on TAKAY medium compared to other four different fungal media since TAKAY medium consists of many nutrients and compounds [20].
In our study, an increasing trend of mycelial growth of the fungus was observed up to 25˚C temperature and started to decline afterward (Figure 3(B)). Notably, there was no mycelial growth recorded at 35˚C temperature. Current findings conform to the results of other investigators who observed the mycelial growth of three species of Pestalotiopsis (Pestalotiopsis fici, Pestalotiopsis guepinii and Pestalotiopsis palmarum) was attained a maximum of 94% of the petri plates after five days of incubation [18] [21]. Likewise, Pestalotiopsisfunerea grew the maximum at 25˚C [22]. Moreover, the growth rate of Pestalotiopsismicrospora was found to be increased with temperature and attained their optimum at 23˚C [19]. Furthermore, it was reported that the temperature ranges between 22˚C - 28˚C were optimum for the mycelial growth of all isolates of P. microspora [18]. Importantly, P. theae did not grow at higher temperatures (35˚C) [23]. Similar results were also reported by other researchers who found that Pestalotiopsismicrospora failed to grow at 33˚C [19] and Pestalotiopsis at 35˚C [23]. The mechanism could be due to the inactivation of key enzymes of the metabolic pathway by elevated temperature [24] [25].
The light was found as an essential determinant for the vegetative growth and development of fungi. The growth rate of P. theae was the maximum under complete dark conditions, surprisingly there was no mycelial growth of the fungus found under continuous light conditions (Figure 3(C)). Importantly, the studied fungus’s fluffy growth was observed under complete dark conditions; however, there was no sign of sporulation under this condition. Besides, the mycelial growth rate was comparatively slower although a significant amount of spore formation was noticed under 12h/12h alternate light and dark conditions. Our results are partially agreed with the earlier works of [18], who reported the best growth of fungi (Pestalotiopsis fici, Pestalotiopsis guepinii and Pestalotiopsis palmarum) under the dark condition; although alternating 12 hours light plus
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Figure 3. Effect of fungal culture media, temperature and pH on mycelial growth of P. theae after 7 dpi. Data represents mean ± standard error of six replicates.
12 hours darkness produced alternative rings between fluffy and abundant in cultures growth. In another lab bioassay, Pestalotiopsis microspora was isolated from diseased tissues of oil palm and incubated on PDA at 25˚C under dark conditions [18] [21].
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Figure 4. Effect of bio-control agents and fungicides on mycelial growth of P. theae at 7 dpi. Here, T1—T. reesei; T2—T. harzianum; T3—T. asperellum; and T4—Amistar Top 250 ppm; T5—Amistar Top 500 ppm; T6—Amistar Top 750 ppm; T7—Ridomyl Gold 250 ppm; T8—Ridomyl Gold 500 ppm; T9—Ridomyl Gold 750 ppm; T10—Tilt 250EC 100 ppm; T11—Tilt 250EC 250 ppm; T12—Tilt 250EC 500 ppm. (A)T. reesei vs P. theae; (B) T. harzianum vs P. theae; (C) T. asperellum vs P. theae; (D) Control (P. theae); (E) Amistar Top 250 ppm; (F) Amistar Top 500 ppm; (G) Amistar Top 750 ppm; (H) Control (P. theae); (I) Ridomyl Gold 250 ppm; (J) Ridomyl Gold 500 ppm; (K) Ridomyl Gold 750 ppm; (L) Control (P. theae); (M) Tilt 250EC 100 ppm; (N) Tilt 250EC 250 ppm; (O) Tilt 250EC 500 ppm; and P: Control (P. theae).
3.3. Effect of Bio-Control Agents and Fungicides on the Targeted Fungus
In the current study, very promising results were found concerning the restriction of the vegetative growth of the targeted fungus by bio-control agents (Figure 4). Different bio-control agents were showed a varying degree of growth inhibition. Fungal pathogen—P. theae was completely inhibited by T. asperellum, followed by 70% mycelial inhibition was found due to T. reesei and the least 50% inhibition of vegetative growth was observed by T. harzianum. In a lab bioassay, it was reported that bio-control agents can produce water-soluble and volatile metabolites and act against soil-borne fungal pathogens [26] [27]. Trichoderma spp. was found as an excellent bio-control agent against fungal pathogens [16]. Furthermore, a group of researchers obtained the promising inhibition of mycelial growth of Fusarium solani by the bio-control agents Trichoderma spp. [12].
In the present investigation, Ridomil gold (Chemical group: 64% Mancozeb 4% Metalaxyl-M) gave an excellent mycelial growth inhibition of the targeted fungus and completely inhibited the mycelial growth of P. theae in all concentrations(Figure 4). Like Ridomil gold, fungicide-Tilt 250EC (Chemical group: Propiconazole) showed the best inhibition of mycelial growth of the targeted fungus, where P. theae was completely inhibited by all of the three concentrations (100 ppm, 250 ppm and 500 ppm). Although the lower concentrations (250 ppm and 500 ppm) of Amistar top (Chemical group: Azoxystrobin + Difenoconazole) inhibited the mycelial growth of the fungus over 80%. Importantly, the higher concentration (750 ppm) of Amistar top could restrict the mycelial growth of P. theae completely. Our results conform to the findings of other researchers who reported propiconazole as an effective fungicide against Pestalotiopsis guepinii [28] [29]. It has been reported that fungicides Tilt 250EC and Amistar Top 325 SC were effective against the mycelium growth of Curvularialunata [16]. Similarly, scientists have evaluated several concentrations of Ridomyl Gold MZ 68 Wg, Tilt 250 EC and Amistar Top 325 SC fungicides where Tilt 250 EC was effective against the mycelium growth of Fusariumsolani [12].
Acknowledgments
We greatly acknowledge the financial support provided by Jahangirnagar University Research grant. We are grateful to Biotechnology and Genetic Engineering Department, Jahangirnagar University for their technical support during the investigation.