The First Report of Fusarium sacchari Causing Yellow Leaf Spot Disease on Rhynchostylis gigantea Orchids in Thailand

Corresponding Author: Krieng Kanchanawatee, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; Email: Krieng.K@chula.ac.th Abstract: Yellow leaf spot disease was observed on Rhynchostylis gigantea orchids on an orchid farm in the Nonthaburi province of Thailand. Symptoms started with yellowish patches lacking definitive borders on the leaves of the orchids. The patches slowly expanded and merged, creating larger patches; a brown spot or black necrotic area then emerged at the center of some of the patches. R. gigantea leaves exhibiting these symptoms were used as the source for isolation studies. Based on the morphological characteristics of the fungal isolate (colony appearance, macroconidia, microconidia, conidiophore and chlamydospore structures), phylogenetic analysis of translation elongation factor-1α sequence and fulfillment of Koch’s postulates, the causative pathogen was identified as Fusarium sacchari. To our knowledge, this study is the first to report F. sacchari as a pathogen of R. gigantea.


Introduction
Orchids are the most exported tropical ornamental crop of Thailand. Among the ornamental orchids, the genus Rhynchostylis is one of the most popular cultivations due to its unique fragrance and long tapering inflorescences (Thammasiri, 2016). However, fungal infestation is a major problem in cultivated Rhynchostylis species. This led us to survey fungal diseases on orchid farms in the central region of Thailand. On an orchid farm in Nonthaburi province, Thailand, we found many Rhynchostylis gigantea plants damaged by yellow leaf spot disease, leading to progressive death from the infected area.
Fusarium species can be identified by their macroscopic and microscopic morphological characteristics, colony appearance, macroconidia and microconidia structures, conidiophore structures and chlamydospores (Leslie and Summerell, 2006). However, since many Fusarium belong to a species complex, accurate identification based solely on morphology is not reliable due to the difficulty in distinguishing between species (Omar et al., 2018). Molecular identification is an additional tool for Fusarium identification that can be used with morphology. DNA sequence analysis of the translation elongation factor-1α (TEF-1α) gene has been successfully used to identify Fusarium species (Geiser et al., 2004;O'Donnell et al., 1998).
Although Fusarium species are known pathogens of orchids, knowledge of the species-specific interactions between host orchids and pathogenic fungi is still limited. The present study was conducted to identify Fusarium fungi isolated from R. gigantea exhibiting yellow leaf spot disease.

Sampling and Fungal Isolation
Four infected R. gigantea plants were collected from an orchid farm in Nonthaburi province, Thailand. The plants were brought to the laboratory where the infected leaves were cut. The fungus isolation was performed from the infected young leaves. Leaves were washed three times with detergent and transferred to 20% (v/v) household bleach (Clorox) and 0.1% (v/v) Tween 80 for 20 min. The leaves were further washed with 70% (v/v) ethanol for 5 min and finally rinsed with sterile deionized water. To verify that the surface of the leaves was completely sterile, the deionized water used for rinsing the leaves in the final step was plated onto Luria-Bertani agar medium (HiMedia, India) and potato dextrose agar medium (PDA, HiMedia, India) and cultured at 27°C for 7-10 d to check for bacterial and fungal colonies.
The surface-sterilized orchid leaves were cut into 11 cm squares and then placed on the PDA plates and incubated at 27°C until mycelia appeared. Singlespore cultures were established using the dilution plating technique (Hansen and Smith, 1932) and then used in all experiments.

Morphological Identification
To assess macroscopic characteristics, fungi were cultured on PDA at 27°C for 7-10 d. The colors of the colonies on both sides of the petri dishes were determined according to Summerell et al. (2003). The color and appearance of the sporodochia from a culture grown on Carnation Leaf Agar (CLA) at 27°C for at least 2 weeks was also observed (Leslie and Summerell, 2006). CLA was prepared in accordance with Leslie and Summerell (2006). To assess microscopic characteristics, observations were performed on the 2-to 4-week-old cultures grown on CLA at 27°C (Hafizi et al., 2013). The appearance, width and length of macroconidia and microconidia were recorded using a compound light microscope with a digital camera. Conidiophores (monoor poly-phialides) were also observed (Summerell et al., 2003) using the slide culture techniques in accordance with Johnson (1946). Briefly, 1.01.0 cm blocks of PDA were placed onto a sterile slide. The fungus was then inoculated at the margin of the PDA blocks with a sterile loop, followed by placement of a sterile coverslip over the block and incubation at 27°C for 3 d. After incubation, slide cultures were stained with lactophenol cotton blue and observed under a compound light microscope with a digital camera. All morphological characteristics were observed in the fungal cultures grown under a 12 h alternating period of light and dark.

Fungal DNA Extraction
DNA extraction was performed according to Cenis (1992) with minor modifications, where 400 μL extraction buffer (200 mM Tris pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% (w/v) sodium dodecyl sulfate) was added to a loop-full of mycelia. The mycelia were ground using a plastic pestle in a 1.5-mL microcentrifuge tube and 8 μL of 10 mg/mL RNase A was added to the suspension and incubated at 65C for 10 min. Then, 150 μL of 3 M sodium acetate (pH 5.2) was added and incubated at -20C for 10 min, followed by centrifugation at 10,000 x g for 15 min. The supernatant was transferred to a new 1.5-mL microcentrifuge tube and 650 μL of isopropanol was added, mixed and centrifuged at 10,000 x g for 10 min to precipitate the DNA. The DNA pellet was washed with 70% (v/v) ethanol, air-dried, dissolved in 100 μL of dH2O and kept at -20C until used.

PCR Amplification and DNA Sequencing
For fungal identification, the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and TEF1-α gene of DNA were PCR-amplified and sequenced. The ITS1-5.8S-ITS2 region was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers (White et al., 1990) with thermal cycling at 95C for 1 min followed by 30 cycles of 95C for 30 s, 57C for 30 s and 72C for 50 s and then a final 72C for 10 min. The TEF-1α region was PCR-amplified using EF-1 (5'-GTTAAGAGGCGCGGTGTCGGTGTG-3') and EF-2 (5'-GGAAGTACCAGTGATCATGTT-3') primers (O'Donnell et al., 1998) with thermal cycling at 95C for 1 min, followed by 30 cycles of 95C for 30 s, 53C for 30 s and 72C for 50 s and then a final 72C for 10 min. The DNA fragments were amplified using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) thermocycler. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Germany) according to the manufacturer's protocol and sent for commercial Sanger sequencing at AITbiotech, Singapore. Sequencing was performed on both strands using the same forward and reverse primers used for PCR amplification.

Phylogenetic Analysis of the Sequence Data
The ITS1-5.8S-ITS2 nucleotide sequences were compared to sequences in the GenBank database using the BLASTn algorithm (http://blast.ncbi.nlm.nih.gov/; Clark et al., 2016). The TEF-1α nucleotide sequences of species related to the sample and outgroup were selected from the FUSARIUM-ID database to construct phylogenetic trees (Geiser et al., 2004). Sequences were aligned by CLUSTAL W using multiple alignment in the Molecular Evolutionary Genetics Analysis 7 (MEGA 7) software (Kumar et al., 2016). The distance of relationships among these same pathogens was constructed using the distance-based Neighbor-Joining (NJ) algorithm with Kimura's two-parameter calculation model in MEGA 7. For statistical support, a bootstrap of 1,000 times was implemented.

Detached Leaf Inoculations
Detached leaf inoculations were performed on fullydeveloped R. gigantea leaves. The leaves were washed five times with tap water followed by 70% (v/v) ethanol and sterile distilled water. Cotton wool soaked with sterile water was used to provide water and a moist environment for the leaves. Mycelium plugs cut from the edge of growing colonies on PDA were placed on the adaxial surface. For wounded inoculations, the leaves were wounded by a sterile needle before placing the mycelium plugs at the injury locations. The inoculated leaves were kept in sealed clear plastic containers and held at 100% RH and 28C with a 18 h photoperiod for 14 d. The cotton wool was changed every 3 d.

Morphological Identification and Characterization of Pathogens
The diseased R. gigantea used in this study was selected from an orchid farm in Nonthaburi province, Thailand, where the R. gigantea were heavily infected with disease symptoms resembling yellow leaf spot disease on leaves. The symptoms started with 1-2 mm yellowish patches without definitive borders. The patches then slowly enlarged and merged with adjacent patches. A brown spot or black necrotic area then develop at the center of some of the yellowish patches while the disease slowly spread to the entire plant ( Fig.  1A and 2A). Late infection turned whole leaves brown and wilted. The leaves still clung to the stem and were ready to drop off (Fig. 1B).
With regard to the macroscopic characteristics of fungal morphology, the colony color of the PDA culture was initially orange-yellow and became purple-white when aged ( Fig. 2B and 2C). When the fungus was cultured on CLA, the sporodochia had a cream color and globular shape (Fig. 2D). With regard to microscopic characteristics, macroconidia production was largely induced on CLA. The morphology of the macroconidia was slender, slightly falcate and thin-walled. Although three septate macroconidia were the most common, two and four septate macroconidia were also observed. The mean length and width of macroconidia was 47.38±4.8 μm and 2.58±0.3 μm (N = 50), respectively (Fig. 2E). Macroconidia were also observed when the fungus was cultured on PDA, but at a very low frequency. The fungus grown on CLA or PDA produced abundant microconidia in the aerial mycelia. The shape of the microconidia was oval and slender with one or no septa. The mean length and width of microconidia were 8.85±2.3 μm and 1.74±0.3 μm (N = 50), respectively (Fig. 2F). The microconidia were produced on short mono-or polyphialides in false heads ( Fig. 2G and 2H).
Chlamydospores were rarely found in cultures grown on PDA. Single verrucose chlamydospores were detected in slide cultures grown on PDA at 3 d after inoculation (Fig. 2I). Morphological characteristics of the fungus were consistent with the description of Fusarium sacchari, which is a member of the Gibberella fujikuroi species complex (Leslie and Summerell, 2006).

Molecular Identification and Phylogenetic Analysis
The BLASTn analysis of the ITS1-5.8S-ITS2 sequence confirmed that the fungal isolate was in the G. fujikuroi species complex (the closest species with 100% identity were F. sacchari and F. verticillioides), while the clean sequence suggested the presence of a single fungal isolate, or at least a single common isolate. The resulting ITS1-5.8S-ITS2 sequence was submitted to GenBank (accession No. MH752426). To construct phylogenetic relationships, the TEF-1α sequences of 30 reference strains and the outgroup F. solani (NRRL 22157) were retrieved from the FUSARIUM-ID database. The TEF-1α sequence of the present fungal isolate was submitted to GenBank (accession No. MH756646). The NJ phylogenetic tree showed that the fungal isolate was grouped in the same clade with the F. sacchari reference strain with a bootstrap value of 100% (Fig. 3). Thus, the combined phylogenetic and morphological data verified the identity of the isolate as F. sacchari strain KC8.

Detached Leaf Pathogenicity Test
The detached leaves were used to confirm the ability of F. sacchari strain KC8 to infect the leaves of R. gigantea. At 14 d post-inoculation, necrotic spots with yellow halos appeared on the adaxial surface of the wounded leaf. On the abaxial surface of the wounded leaf, yellow spots appeared which somewhat resembled the yellow spots on the adaxial surface of the unwounded leaf, but with stronger disease symptoms. No symptoms were observed on the wounded and unwounded leaves without mycelium plugs (Fig. 4). The re-isolated fungus from the inoculated leaves was morphologically identical to the F. sacchari in the present study, therefore fulfilling Koch's postulates.

Discussion
Fusarium sacchari is an economically important plant pathogen of sorghum (Leslie et al., 2005), wheat (Wang et al., 2014), maize (Hsuan et al., 2011;Leslie et al., 2005), banana (Tarnowski et al., 2010), rice (Hsuan et al., 2011;Petrovic et al., 2013) and sugarcane (Egan et al., 1997;Hsuan et al., 2011;Poongothai et al., 2014;Viswanathan et al., 2017;. In the present study, F. sacchari was isolated from R. gigantea leaves with yellow leaf spot disease, adding R. gigantea orchids to the host list of F. sacchari. The symptoms of the disease were somewhat similar to the yellow leaf spot disease observed in Cymbidium orchids infected with F. proliferatum and F. subglutinans (Ichikawa and Aoki, 2000), except the brown areas and black necrotic areas did not always develop. However, the black necrotic areas were observed on the detached leaves of R. gigantea at wounded inoculation sites (Fig. 4).   (Leslie and Summerell, 2006). However, the sporodochia color and the presence of chlamydospores disagreed with the manual's descriptions. The color of sporodochia in the present study was cream instead of orange. This might be due to the variation within the species, as observed in the Fusarium oxysporum sporodochia that may appear from cream to orange (Beale and Pitt, 1989;Smith et al., 1988). In contrast to the absence of chlamydospores described in The Fusarium Laboratory Manual, we found single verrucose chlamydospores produced on the third day after inoculation onto PDA. This has also been reported previously (Poongothai et al., 2014), where three F. sacchari isolates out of 117 formed chlamydospores on PDA medium.
The fulfillment of Koch's postulates confirms that F. sacchari strain KC8 is the cause of yellow spot disease on R. gigantea. F. sacchari strain KC8 was able to infect both wounded and unwounded leaves, indicating pathogenic ability of the fungus to penetrate through the surface of the plant. Although the re-isolated fungi had all morphological characters resembling the inoculated F. sacchari strain KC8, the molecular characterization of TEF1-α gene should also be performed to confirm identification.
This present study not only provides information that can be applied to the disease management of R. gigantea orchids, but also serves as a precaution to protect persons in contact with the disease, as F. sacchari can cause mycotic keratitis and exogenous fungal endophthalmitis in non-immunocompromised farmers (Bansal et al., 2016;Chander et al., 2011).

Conclusion
This is the first report of F. sacchari causing yellow leaf spots on R. gigantea orchids, as supported by morphological characteristics, phylogenetic analysis of the TEF1-α gene and the fulfillment of Koch's postulates. Symptoms of the disease and the pathogen causing the disease are crucial to establish a disease management program, as this disease leads to the death of infected orchids.