STUDIES ON VASCULAR INFECTION OF FUSARIUM OXYSPORUM F. SP. CUBENSE RACE 4 IN BANANA BY FIELD SURVEY AND GREEN FLUORESCENT PROTEIN REPORTER

Fusarium wilt of banana (Musa spp.) caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most serious banana fungal diseases in the world. Understanding the infection process of Foc is important for development of effective ways in disease control. In order to follow infection and colonization of this pathogen from root to rhizome and pseudostem tissues of banana, a highly pathogenic strain FJAT-3076 of Foc race 4 (Foc4) was transformed with gene encoding green fluorescent protein (GFP) and the fungus carrying gfp (FJAT-3076-GFP) was used to inoculate banana plants (Cavendish cv. B.F.). After inoculation for 3 to 10 d, it was observed that the conidia and their germtubes had penetrated into epidermis of young roots. The hyphae were found inside the root xylem 10 d after inoculation in the rhizome and pseudostem xylem after inoculation for 17 d. All plants infected by Foc died in 24 d after inoculation. It was also observed that Foc had spread all over the xylem and part of hyphae reached the pseudostem surface. Hyphal population was found the highest in the pseudostem, lower in root and least in rhizome. Field survey confirmed that Foc4 were mostly present in the base of pseudostem and less in the rhizome. Thus, effective prevention of the Foc hyphae movement from the rhizome up to the pseudostem might delay or control banana wilt disease.


INTRODUCTION
Fusarium wilt of banana, commonly known as Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most serious fungal diseases in banana, and a major limiting factor of banana production worldwide (Getha & Vikineswary 2002). Foc is divided into four races based on host susceptibility. For example, Cavendish cultivars (Musa spp. AAA-group), the most commonly planted banana in production area, are highly susceptible to Foc race 4 (Foc4). The fungus can survive as a saprophyte for numerous years. Once presenting in the soil, Foc is not able to be eliminated (Kurtz & Schouten 2009). It initially infects the roots of banana plants, then colonizes the vascular system of the rhizomes and pseudostems, eventually leads to typical wilt symptoms, including foliage chlorosis and necrosis before the plant died. To date, few options are available in protecting banana from Fusarium wilt disease (Forsyth et al. 2006). Recently, the application of transformed strains expressing reporter genes and facilitated observation on the development of pathogens within their plant hosts (Paparu et al. 2009). In the study of Fusarium wilt of banana, Visser et al. (2004) had transformed Foc4 with gfp gene, and observed the fluoresced fungal hyphae within tissues of infected plants. Li et al. (2011) developed a GFP-tagged Foc4 transformant and studied the pathogenesis using both fluorescence microscopy and confocal laser scanning microscopy (CLSM). They found that fungal hyphae were able to penetrate cell walls directly to grow inside and outside cells in banana roots, and fungal spores were produced in the root systems and rhizomes. However, how Foc4 progresses in the vascular tissue of banana roots to the pseudostem, which correlates with Foc pathogenicity, is not clearly known. In this study, we report the observation of Foc4 systemic infection process in a Cavendish banana with GFP reporter, mainly focus on Foc4 movement from roots to pseudostem. More understanding the infection process of Foc4 is required and helpful for development of banana Fusarium wilt control.

Isolation, identification and distribution of Foc4 in field banana plants:
Three banana plantations of Cavendish cultivar were chosen for experimental sites at Zhangzhou Country, one of the main bananaproducing regions in South of China (Longitude 117.37, Latitude 24.52). A symptomatic plant with yellowing and wilting older leaves and an asymptomatic plant were gathered from each experimental site. Twenty grams of tissue samples were obtained from the different positions of banana plant as follow: root, rhizome, base pseudostem, middle pseudostem and top pseudostem; and they were marked as sample I, II, III, IV and V in turn. The samples were surface disinfected with 10% sodium hypochlorite and homogenized with sterile distilled water. Dilution series were made of each suspension and plated onto Komada medium for isolation of F. oxysporum (Komada 1975). For the pathogen distribution inside banana plant, the singlespore isolate from each sample was numbered and the average fungal contents at the same position of three symptomatic or asymptomatic plants were calculated. For phytopathogen identification, 30 single-spore colonies from each sample at different positions of banana were chosen. The mycelia of single-spore isolates were transferred to fresh potato dextrose agar (PDA) medium for 7 d, and then scraped directly from agar plates and used for DNA isolation. Total genomic DNA was extracted according to fungal DNA kit (Omega Bio-tek, Norcross, Georgia). The specific PCR for F. oxysporum and Foc4 were detected according to Nel et al. (2006) and Lin et al. (2010), respectively.

Foc race 4 strain and gfp gene transformation:
The Foc race 4 isolate, FJAT-3076, isolated from pseudostem of a diseased banana in this study was selected for the GFP transformation. The fungus was tested for its aggressiveness on Cavendish banana cv. B.F., resulting in the wilting of 100%. Strain FJAT-3076 was modified with the gfp gene using the sGFP expression vector pCT74 according to Lorang et al. (2001). Fungal protoplasts of FJAT-3076 were transformed using a polyethylene glycol/CaCl2mediated transformation method as described by Liat et al. (2003). The colony with the highest GFP expression was chosen under a fluorescence stereomicroscope. The mycelia with bright green fluorescence were submitted to six successive subcultures on PDA without antibiotic, in order to obtain stable transformants (Sarrocco et al. 2007). Growth characteristics and pathogenicity of GFPexpressing isolates of Foc were verified using the inoculation protocol described by Xiao et al. (2009).

Banana plant inoculation by Foc transformants:
A Foc transformant with the highest GFP expression, FJAT-3076-T2, and the wild-type Foc strain FJAT-3076 were chosen for inoculation. The fungi were cultured respectively into 500 mL potato-dextrose broth (PDB) in 2 L erlenmeyer flasks on a shaker at 170 rpm min -1 . After 5 d, the conidial suspension was passed through sterile cheesecloth to separate the mycelia from the conidia and adjusted to a final concentration of 10 6 spore mL -1 . Due to the high susceptibility to Foc, Cavendish banana cv. B.F. was chosen for plant inoculation. Thirty plants were used for the inoculation trial per strain. Banana cuttings (50-d-old tissuecultured plant) were inoculated by root dipping in the spore suspensions for 30 min. Two plants per pot (17.0 cm × 17.0 cm × 12.0 cm) were ultimately retained in a greenhouse at 28 °C, 80% relativity humidity and 12 h light/dark. Control plants were treated with sterile water and maintained in the similar situation.

Vascular colonization of banana plant by Foc examined or viewed under CLSM:
Following symptom appearance at different phases, microscope observations of Foc colonization in banana plant were performed at 3, 10, 17 and 24 d after inoculation. Inoculated and control plants were carefully taken out of the pot and gently rinsed in tap water to wash away soil particles and unattached fungi. Longitudinal sections of root, rhizome, pseudostem and leaf were hand-sectioned and each placed directly on a glass slide in a water drop with a cover slip. Above 50 sections from different positions were viewed by microscope. The microscopic examinations were performed by using a Leica SP5 confocal laser scanning microscope (Wetzlar, Germany) with emitted light at 500-525 nm.

Identification and distribution of Foc4 in field banana:
The Komada medium was used to isolate F. oxysporum from banana plants at root, rhizome, base pseudostem, middle pseudostem and top pseudostem. The Fusarium colonies were obtained only from symptomatic plants; none was found in the asymptomatic plants. All the isolated Fusarium spp. were identified as F. oxysporum by FOF1/FOR1 primer set with the amplification of a single 340-bp DNA fragments. Among which, all the isolates were also confirmed as Foc4 identified by the Foc1/Foc2 primer set, except 40% of the isolates from root were not Foc4 ( Fig. 1 and Table 1). Moreover, there were significant differences among contents of Foc4 at different positions of diseased bananas. The fungi existed mostly at base pseudostem with an average concentration of 10.4 ×10 2 colony forming unit (cfu) g -1 , following with at middle pseudostem (4.76×10 2 cfu g -1 ), root (1.02×10 2 cfu g -1 ), top pseudostem (0.98×10 2 cfu g -1 ) and rhizome (0.24×10 2 cfu g -1 ). Therefore, Foc4 existed least in the rhizome, only 23.5 and 2.3% of those in the root and the base pseudostem.

bp
Green fluorescent colonies, arising from the regenerated protoplasts, became visible at 5 d after transformation. Those putative transformants with highest GFP expression were chosen under a fluorescence stereomicroscope (Fig. 2a) and transferred to six successive subcultures on PDA without antibiotic. Strong constitutive expression of sGFP occurred and could be visualized in fungal hyphae, microconidia, macroconidia and conidiogenous cells (Fig. 2b-2d). Neither virulence nor morphological characteristics (growth rate, spores or hyphae) of the pathogen was altered by transformation with the plasmid pCT74. A Foc transformant with the highest GFP expression, FJAT-3076-T2, was chosen for further study.

Vascular colonization of banana plant by Foc examined or viewed under CLSM:
In the plant inoculation tests, Foc4 transformant FJAT-3076-T2 was as pathogenic as the wild-type strain FJAT-3076, causing 100% mortality rate of banana plants tested at 24 d after inoculation. In order to study the colonization of banana plant by Foc4, transformed strain FJAT-3076-T2 was microscopically followed in whole plant and in longitudinal sections from 3 to 24 d after inoculation by CLSM. Inoculated for 3-10 d, the pseudostems and leaves of plant were symptom-free, but most roots performed severe yellowing and subsequent discolored. The conidia or their germ-tubes of Foc4 were observed to penetrate epidermal cells of young roots (Fig. 3a-b) and the hyphae developed in the root xylem (Fig. 3c). However, the green hypha couldn't be visible except root tissue. By 17 d, the wilt symptoms became visible on banana plants inoculated with both transformants and wild-type isolate. The leaves at the bottom appeared yellowing and one half of the rhizome was discolored (Fig. 4). Hyphae were visible in the root, rhizome and pseudostem. On the 24 th day after inoculation, the plant became dying and three quarters of the rhizome were discolored. The green-fluorescing b d a c hyphae were visible in the root, rhizome and pseudostem, but not in the leave. Some hyphae reached the pseudostem tissue and were very obvious intercellular growth in these tissues (Fig. 3d). Parts of hyphae were confined in xylem vessel and grew upwardly (Fig. 3e). A few of conidia could be also visible within the root or pseudostem vessels tissues of banana during the course of infection (Fig. 3f).  Furthermore, obvious differences were also figured out among the Foc4 densities at different plant positions (root, rhizome and pseudostem) after inoculation for 24 d. The colonization on the root developed continuously; small hyphal networks were observed within the root tissues by CLSM (Fig. 5a). Only a few hyphae were observed in the rhizome tissues (Fig. 5b), whereas denser hyphal network presented on the surface and inside the pseudostem tissues (Fig. 5c). Therefore, the highest level of hyphal density was in the pseudostem, whereas the level was much less in root and only a few hyphae were found in rhizome.

DISCUSSION
Using GFP-expressing strains to monitor invasion and infection, the plant infection by F. oxysporum was found to be a complex process, which comprised several stages of host-pathogen interaction: recognition of the host roots and adsorption, penetration of hyphae through the different root tissues, penetration and progression in the xylem, and adaptation to the internal plant environment. To be successful, the fungus should overcome different plant defence responses at each stage (Di Pietro et al. 2003;Michielse & Rep 2009;Zvirin et al. 2010). For example, Lagopodi et al. (2002) visualized the F. oxysporum f. sp. radicis-lycopersici (Forl) invasion of a susceptible tomato cultivar at 1-3 day after inoculation.
Forl attached itself to root hairs by means of fungal mycelia and penetrated portions of the root at random, followed by penetration along epidermal cell borders after 4 d. Zvirin et al. (2010) mentioned the attachment of F. oxysporum f. sp. melonis (Fom) to a susceptible melon cultivar had occurred along the root epidermis at 3 th day after inoculation. Fom was discovered in the root xylem by day 4, and then the xylem of the root and hypocotyl were heavily populated with mycelia by day 11 and all seedlings were dead by day 14. The conidia were produced and germinated within the vessels. For infection of banana with Foc, Li et al. (2011)

b C a
Foc was visible within the root vascular cylinde tissues of banana by day 10. The hyphae were up to the rhizome and pseudostem xylem by day 17. All plants were dead at 24 d after inoculation; whilst Foc had spread all over the xylem and part of hyphae reached the pseudostem surface. The conidia of Foc were also visible at different positions of banana tissues during the course of infection. It could be explained that sporulation and germination of secondary mycelium was crucial for rapid upward colonization (Beckman & Roberts 1995). An interesting finding in this study was the obvious differences among Foc populations at different banana positions during the infection phase, both in field plants by pure culture method and in laboratory plantlets under CLSM examination. In symptomatic banana plants collected from field, Foc4 was found to exist least in the rhizome, only 23.5% of those in the root and 2.3% of those in the base pseudostem. Microscopic examination of banana plantlets inoculated with virulent Foc4 showed that the highest level of hyphal density was observed in the pseudostem, while root had middle level and rhizome had the least. Sampled equally for about 2 × 2 cm size, few of hyphal pieces could be found in the rhizome tissues, whereas a mass of hyphae could be discovered within the root and pseudostem xylem. It was speculated that the hyphae were blocked or delayed to progress within the rhizome xylem, which could be related with the banana's histophysiological characteristic. The banana plant was a giant perennial herb that consisted of a rhizome and a pseudostem (Robinson 1996). The rhizome consisted of starchy parenchyma cell, and was a nutrients store house that could transfer all the nutrients stored thereto the growing suckers (Wu 2006;Thakorlal et al. 2010). Differently, the pseudostem was composed of several tightly packed leaf bases that consisted of aerenchymas in the parenchyma cell (Wu 2006). The plant, moreover, was often able to prevent infection from successfully travelling to and entering the rhizome by the production of gels and tyloses (a resistance mechanism) to seal off the infection (Daly & Walduck 2006). But, if few of hyphae progressed into the pseudostem through the rhizome, the hyphae would spread rapidly in the aerenchymas and travel all the way up to the top of the pseudostem. Eventually, the pseudostem of the plant was easily damaged and died.
Foc was difficult to be controlled using fungicides and be eradicated from the soil using fumigants (Daly & Walduck 2006). However, Foc might be delayed or controlled, if it was effectively preventing the hyphae from the rhizome up to the pseudostem, such as injecting fungicide at the points above the rhizome.