Morphological, phylogenetic and pathogenicity characterisation of Fusarium species associated with wilt disease of pumpkin (Cucurbita pepo Linnaeus)

Fusarium is a well-known soil-borne fungus where most species belonged in this genus is prominently phytopathogenic. Nevertheless, this pathogenic species has affected the production of pumpkin worldwide. This study underlines the morphological, phylogeny and pathogenicity characteristics of Fusarium for a better disease-control strategy. Twenty-six Fusarium isolates were collected from wilt infected pumpkin in various locations of Peninsular Malaysia. From the combinations of morphological and molecular identifications, four species were identified as F. oxysporum (2 isolates), F. solani (4 isolates), F. proliferatum (7 isolates) and F. incarnatum (13 isolates). Microscopic and macroscopic observation visualized distinct characteristics of the identified Fusarium species. Sequence analyses of tef1α and β-tub genes inferred by maximum likelihood tree resulted in distinct section-specific characteristics. Meanwhile, pathogenicity test of Fusarium isolates presented by the seed inoculation produced various degrees of severities. Fusarium solani C2526P recorded the highest severity of 93.8% after 30 days of post inoculation (dpi). Symptoms have been identified as early as 10 dpi producing stunted growth of the plants. On the other hand, Fusarium oxysporum D2532P recorded 85.3% disease severity. Pathogenic Fusarium caused stunted growth, chlorosis, wilting and necrosis especially at the root of pumpkin plants. This study provides valuable information and methods to manage wilt infected pumpkin in the future.

Commonly, pumpkin is consumed as food and applied in ethno-medicinal applications in several countries such as China, Mexico and India (Alarcon-Aguilar et al., 2002;Aggarwal and Kotwal, 2009). However, this highly demanded crop is being attacked by various fungal infections mainly in the field. Fusarium wilt disease is recognized as a yield-limiting factor in various cucurbit productions. This disease can be perceptible by stunting, yellowing of lower leaves, progressing wilting, defoliation, necrosis of the vascular tissue and death of plants (Chehri et al., 2011;Caroline and Olubukola, 2013;Redda et al., 2018). The most important pathogens causing the disease are Fusarium oxysporum and Fusarium solani. These pathogens could survive in the soil for several years in a form of chlamydospores (Callagan et al., 2016) and have become the main limiting factor in managing disease dissemination. Thus, characterization of pathogenic Fusarium is essential for integrating disease management to limit the extension of its host range. Several pathogenic species in the genus Fusarium are specialized with respect to their host specificity formerly known as formae speciales (f. sp.) (Snyder and Hansen, 1940). This specialization was recognized as the physiological capabilities of Fusarium strains onto one or a few species of plant. The pathogenic species is considered arduous to be controlled due to several aspects such as its ability to produce resistance structures, resistance to fungicides, manipulation on host defence responses and the ability to produce mycotoxins (Van Dam et al., 2016;Moreno-Velandia, et al., 2019). However, several species belong in the genus were considered saprophytic while others pathogenic. Identification and recognition of the primary invader could reduce the infection risks. Therefore, the objectives of this study were to isolate and identify Fusarium species associated with wiltinfected pumpkin and to ascertain the pathogenicity test of the isolated Fusarium species.

Fungal isolation
Pumpkin was obtained from five various locations throughout Peninsular Malaysia including Maran and Cameron Highland in Pahang, Tok Bali in Kelantan, Tanjung Karang in Selangor and Tangkak in Johor. Infected leaves and fruits were chosen for fungal isolation and cut into pieces of 1 cm x 1 cm. Infected tissues were soaked in 0.5% sodium hypochlorite (NaOCl) (Chlorox, Oakland, USA) and rinsed twice with sterile distilled water (Liu et al., 2017). Tissues were dried using sterile filter paper and placed on peptone pentachloronitrobenzene agar (PPA) (Sigma-Aldrich, Missouri, USA) followed by incubation for 5 days (Summerell et al., 2003). The cultures were purified by streak plate technique onto potato dextrose agar (PDA) and incubated for 5 days at 28 ± 2 ⁰C (Leslie and Summerell, 2006).

Morphological characterisation
Morphological characterisation was divided into microscopic and macroscopic observations on each isolate. For microscopic examination, cultures were grown on carnation leaf agar (CLA) (Leslie and Summerell, 2006) and incubated for 7 days at 28 ± 2 ⁰C. Matured culture was observed under light microscope CX2Li (Olympus, Tokyo, Japan). With the same observation method, Fusarium cultures grown on water agar (WA) were also examined. Characters such as the conidia size, shape and number of septate, presence of chlamydospore, phialide and hypha were observed and recorded (Leslie and Summerell, 2006). For macroscopic examination, cultures were grown on PDA for 7 days at 28 ± 2 ⁰C. Macroscopic characters included in this study were pigmentation, colony features and presence of sporodochia.

DNA extraction
Fusarium isolates were grown on PDA for 5 days (28 ± 2 ⁰C). With micropipette tip, the mycelia of the culture were scratched prior to DNA extraction. The genomic DNA was extracted by Ultra Clean® Microbial DNA isolation kit (MO-BIO, Carlsbed, CA, USA) according to the procedures by manufacturer.
Nucleotide sequencing and phylogenetic analysis PCR products were submitted to MyTACG Bioscience Company, Malaysia, for purification and sequence analysis. The nucleotide sequence dataset of tef1α and β-tub was aligned by ClustalW using Molecular Evolutionary Genetics Analysis 7.0 (MEGA). Alignments were manually modified to exclude all ambiguous sections from analysis. Basic Local Alignment Search Tool (BLAST) gene polymorphism analysis in comparison with databases from National Centre of Biotechnology Information (NCBI) resulted in related genus species including F. incarnatum NRRL 31160, F. oxysporum NRRL 25369, F. proliferatum NRRL 53578 and F. solani FMR 8021 (Otero-Colina et al., 2010;Azor et al., 2007) as references for the alignment. Aspergillus niger CBS513.88 was used as an outgroup. Maximum likelihood (ML) was inferred to a phylogenetic tree displaying support value of more than 80% (Watanabe et al., 2011).

Conidial suspension
A total of 26 Fusarium isolates were cultured on PDA at 28 ± 2 ⁰C for 5 days prior to inoculum preparation. Matured cultures were added with 10 mL sterile distilled water followed by tender scratch on the filamentous mycelia using sterile micropipette tip. Fungal inoculum of 200 mL was prepared to a final concentration of 2x10 6 conidia/ mL as adopted from Chehri et al. (2011). The mycelial sheets and residues of the media were filtered using sterile cheese cloth and transferred into a 500 mL conical flask.

Seed inoculation
The seeds of pumpkin var. Gold Butter (Green World Genetics Sdn. Bhd., Kuala Lumpur, Malaysia) were sterilised according to Zhang et al. (2012) by soaking into 10% sodium hypochlorite (NaOCl) and rinsed twice with sterile distilled water. Sterilised seeds were then inoculated by soaking into 200 mL fungal conidia suspension for 12 hours in an incubator shaker at 100 rpm in 30±1 ⁰C (Sukanya and Jayalakshmi, 2017). Inoculated seeds were sown into 1 kg soil containing a mixture ratio of (3:2:1 = topsoil: manure: river sand) pre-autoclaved according to Mahmood et al. (2014). Plant treatments were performed in randomised complete design (RCD) in the UPM Glasshouse. Each isolate was prepared with 12 plant replicates and grown within 12/12 hrs at 32±1 °C days and 28±1°C nights with humidity of 72% for 30 days. Plant physiological parameters and disease severity index (DSI) were calculated followed by data collection.

Disease assessment and data analysis
The cultivation was observed for 30 days of post inoculation (dpi). The progress of symptoms appearance was carried out every 5 days. After 30 dpi, the emergence of any particular wilt symptoms was assessed according to disease scale (Table 1) conducted by Schoonhoven and Pastor-Corrales (1994) as well as Raupach et al. (1996) with slight modifications.
The disease severity index was calculated for each isolate according to the parameters in the disease scale (Mwaniki et al., 2011).

Morphological characteristics
A total of 26 isolates were recovered from Maran and Cameron Highland in Pahang, Tok Bali in Kelantan, Tanjung Karang in Selangor and Tangkak in Johor. Five species were identified as F. oxysporum (2 isolates), F. solani (4 isolates), F. proliferatum (7 isolates) and F. incarnatum (13 isolates) based on morphological characteristics according to synoptic keys for species identification. Morphologically, all species presented primary characters, which include macroconidia, microconidia and chlamydospore. All 26 isolates were observed microscopically and macroscopically; this includes size, shape and number of septate of the macroconidia, microconidia and chlamydospore, position of conidia, pigmentation and colony features. Chlamydospores are produced in all Fusarium species colonies except for F. proliferatum (Table 2). All species shared a variety of macroconidia shape with slightly curved and tapered towards each ends. The apical end is elongated especially for F. proliferatum macroconidia. The basal end is where the conidia were attached and it is slightly short. Fusarium oxysporum and F. solani shared similar features of macroconidia, but F. solani has bigger in size. Fusarium proliferatum has the only microconidia observed in chain on CLA as early as 4 days of incubation, whilst other species present detach and individual microconidia. CLA is a substrate medium produced by sterilized carnation leaf-pieces that is particularly useful in uniform conidia identification purpose (Nelson et al., 1983). Among all the species, F. incarnatum colonies grown most rapidly on PDA by displayed thick and cottony aerial mycelia. As the colony grown matured of 5 days old, the pigmentation produced on media could be observed. The colours are initially white and changes as it grown matured. Even though, colonies produced quiet similar pigment but feature and thickness of the aerial mycelia differ among species. The pigmentation produced in the media facilitates morphological distinction between species and served as the initial identification. However, the identification based on morphological characteristic was somehow affected by genotype and environmental conditions, which led to instability and complication in species identification. Thus, the identification of Fusarium could achieve a better accuracy as molecular identification is applied upon morphological speciation.

Phylogenetic analysis
Prior to the construction of presented phylogenetic tree, individual dataset of a single locus was first generated and found to be incongruence to each other. Apart from that, similar dataset constructed did not resolve the high confidence value as high as the stated bootstrap support value. PCR amplification of tef1α and β-tub genes registered a single fragment on agarose gel electrophoresis ranging from 645 to 918 bp and 500 to 693 bp in size ( Figure 1, Table 3).  Based on phylogenetic analysis of combining both genes, four major clades were generated. The first clade comprises four isolates of F. solani. Clade II contains seven isolates of F. proliferatum. Clades III and IV consist of two isolates of F. oxysporum and 13 isolates belong to F. incarnatum, respectively. All isolates belong in the first clade were not diverged in nucleotide substitution represented by the horizontal branch compared to the reference sequence ( Figure 2).

Figure-2: Maximum likelihood trees of Fusarium
isolates and related species inferred from the combination of tef1α and β-tub  This isolate recorded the highest disease severity of 93.8% followed by F. oxysporum D2532P with disease severity of 85.3% (Figure 3). Three of F. proliferatum isolates; B1781P, J1791P and J1793P were identified as moderate virulent. All these three isolates produced average percentages of disease severity of 38.0%, 17.0% and 35.3%, respectively. Fifteen isolates were identified as non-pathogenic with no observable symptoms produced. The symptoms were initially produced by the stunted growth of the plants. Infection starts progressing with the appearance of chlorosis on the lower leaves in which the leaves were wilted and crumpled. Cross section of the primary root structures displayed necrosis after 30 dpi. The lateral roots or the root branches were reduced on the infected plants ( Figure  4) compared to the control plants. Fusarium infection is generally limited to the shoot area, but mainly at the root area. Therefore, severe symptoms can be observed at the root cortex.

Discussion
Molecular identification has provided various dependable outputs of Fusarium identification compared to morphological characterisation. Previously, several markers including protein-coding regions and nuclear ribosomal DNA (rDNA) (Liu et al., 2015;Sanders and Rodriguez, 2016;Kusai et al., 2018;Turrini et al., 2017) were used and resulted in a high quality species identification especially for a closely related species. Protein coding genes have showed a rapid nucleotide substitution rate and subsequently high resolution for closely related species or among conspecific strains (Watanabe et al., 2011). This study presented disagreement of morphological and molecular characterisation, which resulted in opposition to a study on Fusarium species by Trabelsi et al. (2017) in Tunisia. F. solani, F. oxysporum and F. proliferatum have been previously reported to cause wilt disease in most cucurbit plants including pumpkin (Chehri et al., 2011;Najihah et al., 2017;Perez-Hernandez et al., 2017;Rezaee et al., 2018). Once the pathogen is present at the root surface, it penetrates through natural openings and grows into the root cortex. After then, the infection progresses and colonises the xylem vessels. By this stage, it is ready to invade the upper ground structure of the plants (De Sain and Rep, 2015). Fusarium sp. secretes a plethora of effectors that enhance colonisation in xylem vessels. These effectors are named Six (secreted in xylem) proteins that are internalised into plant cells (Francisco et al., 2018). The extension of Fusarium wilt infection caused severe and dead pumpkin plants. The presence of mycelia on the soft infected stem can be observed as a severe symptom of infection. There are several factors that contribute to the severity of Fusarium infection. These include the environmental condition, irrigation (Chehri et al., 2011), climatic conditions (Zhang et al., 2014) temperature and oceanic air (Czembor et al., 2015).

Conclusion
The identification of Fusarium comprising morphological and phylogenetic analyses has revealed better species identification especially for the complex structure of taxonomic like Fusarium. A single piece of infected pumpkin plant has been seen inhabited by various species of Fusarium. These species displayed a wide degree of disease severities. Fusarium solani, F. oxysporum and F. proliferatum are the most important pathogens of wilt disease in pumpkin. From this study, a better Fusarium wilt management could be achieved by accessing the information on the factors affecting the pathogenesis of pathogenic Fusarium. Any biological and physiological factors of pumpkin such as growth rate should be examined upon Fusarium invasion.