Systemic colonization of banana plants by a green fluorescent protein-tagged strain




НазваниеSystemic colonization of banana plants by a green fluorescent protein-tagged strain
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Systemic colonization of banana plants by a green fluorescent protein-tagged strain of Fusarium oxysporum f. sp. cubense


R. F. Xiao, Y. J. Zhu, Y. D. Li & M.X. Su, B. Liu*

Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, PR China

* Corresponding author, e-mail: laeptb@163.com


Abstract: Fusarium wilt of banana (Musa spp.) commonly known as Panama disease caused by Fusarium oxysporum f. sp. cubense. The fungus could infect plant through roots, and eventually blocked the vascular system of the pseudostems, resulting in plant death. In order to reveal the infection and colonization of the pathogen Foc, we studied the distribution of Foc in banana plants from the asymptomatic and symptomatic plants in the filed by pure culture, and located the infection process of Foc by transform Foc isolates with the GFP gene. Foc seemed able to penetrate portions of the root at random by its conidia or germ-tubes when they inoculated for 24 days. After 17 days, the mycelia grew into the rhizome and pseudostem. At inoculated for 24 days, Foc had spread all over the plant and mycelia were up to the pseudostem and formed mycelial networks. Moreover, the similar results were obtained from pure culture and observing by confocal laser scanning microscopy. The highest level of hyphael intensities were observed in the pseudostem, while root had middle level and rhizome had the least.


1 Introduction

Fusarium wilt of banana (Musa spp.), commonly known as Panama disease caused by Fusarium oxysporum f. sp. cubense Snyder and Hanson (Foc) (1940), is one of the most serious fungal diseases in banana, and reported to be one of the major limiting factors for banana production worldwide (Getha and Vikineswary 2002). Evidence suggests that Foc originated in Southeast Asia and from there was disseminated rapidly and affected plantations in almost all banana-growing countries of the world (Koenig et al. 1997). The fungus can survive as a saprophyte for numerous years, start to infect the roots of banana plants, then colonizes the vascular system of the rhizomes and pseudostems, eventually leads to typical wilt symptoms including foliage chlorosis, necrosis before the plant dies. Once Foc is present in the soil, it cannot be eliminated (Kurtz and Schouten 2009). There are four races of the pathogen which are separated based on host susceptibility. Unfortunately, Cavendish cultivars are highly susceptible to Foc race 4, first in the subtropics, and then in the tropics, have raised fears that the world trade in banana might again be threatened (Ploetz 2006). Despite the economic significance of banana, the infection of Foc in banana plants is poorly understood.

To facilitate the observation the development of pathogens within their plant hosts, transformed strains expressing reporter genes have been used, such as the green fluorescent protein (GFP) gene or the red fluorescent protein gene (RFP), have also been used to transform strains of pathogenic fungi and antagonistic bacteria or fungi (Horowitz et al. 2002; Muller-Taubenberger and Anderson 2007; Yan et al. 2007). The GFP has been expressed in numerous filamentous fungi such as Ustilago maydis, Podospora anserina, Magnaporthe grisea, Cochliobolus heterostrophus, Mycosphaerella graminicola, Colletotrichum lindemuthianum, Phytophthora parasitica, Aspergillus niger and F. oxysporum (McLeod et al. 2007; Nahalkova et al. 2008; Visser et al. 2004). In the study of Fusarium wilt of banana, (Visser et al. 2004)transformaed Foc with GFP gene. The fungal hyphae within tissues of infected plants could be seen to fluoresce and the transformed fungus was re-isolated from artificially inoculated plants. (Paparu et al. 2009) studied the plant colonization of endophytic non-pathogenic F. oxysporum isolates in Banana by using GFP marker. However, the infection processes of Foc in detail was remained little known.

The GFP has also been used as a marker gene in many economically important crops in order to visualize the fungi’s colonization and infection behaviors under in vivo conditions. GFP transformation allows the use of a simple fluorescence microscope to trace the growth of Fusarium within host structures and it is a pre-requisite for setting up more sophisticated protocols, such as confocal microscopy, for more detailed study (Sarrocco et al. 2007). Confocal laser scanning microscopy (CLSM),which is an effective, fast, and non-invasive tool allowing spatiotemporal analysis of pathogen-host and fungus-fungus interactions in vivo (Jensen et al. 2001; Sarrocco et al. 2006).

The first objective of the current study was to mark Foc isolate (race 4) with the genes coding for GFP. The second objective was to reveal colonization and location of the pathogen within the banana plant by using the transformed strains as tools under CLSM.


2 Materials and methods

2.1 Identification and distribution of Foc in banana

The banana plants were collected from Zhangzhou country, one of the main banana-producing regions in South of China. Three banana plantations where Fusarium wilt of banana has been found were chosen for experimental sites, in which Williams, the most important Cavendish banana cultivar, were planted.

In order to identify the pathogen of Fusarium wilt and study its distribution in banana plants, a symptomatic plant with yellowing and wilting of the older leaves and an asymptomatic plant which were gathered from every experimental site. Twenty grams of tissue samples were obtained from the different positions of banana plant as follow: root, rhizome, base of pseudostem, middle of pseudostem and top of pseudostem, which 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 (Komada 1975) for isolation of F. oxysporum. For pathogen distribution in plant, the numbers of single-spore isolates from each sample were counted, and the averages of the fungal contents at the same positions of the three symptomatic or asymptomatic plants were calculated.

For the phytopathogen identification, 30 single-spore colonies from each sample position of banana plant were chosen. The mycelia of single-spore isolates were transferred to fresh PDA medium for 7 d, and then scraped directly from agar plates and used for DNA isolation. Dried fungal mycelia were snap frozen in liquid nitrogen and ground to fine powders using a mortar and pestle. Total genomic DNA was extracted according to Fungal DNA kit (OMEGA, USA). Specific PCR for F. oxysporum was detected according to (Nel et al. 2006), a 340-bp size fragment was produced by PCR from DNA using the primer set FOF1/FOR1 (5'-ACATACCACTTGTTGCCTCG/5'-CGCCAATCAATTTGAGGAACG). The parameters for PCR were denatured at 94°C for 60 s, followed by 25 cycles of denaturing at 94°C for 60 s, annealing at 58°C for 30 s, and polymerising at 72°C for 60 s, and with a final extension at 72°C for 7 min. Another specific PCR for Foc race 4 isolates was detected according to (Lin et al. 2010). A 242-bp size fragment was produced by PCR from DNA using the primer set Foc-1/Foc-2 (5'-CAGGGGATGTATGAGGAGGCT/5'-GTGACAGCGTCGTCTAGTTCC). The parameters for PCR were denatured at 94°C for 60 s, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 68°C for 30 s, and polymerising at 72°C for 90 s, and with a final extension at 72°C for 10 min. PCR products were subjected to electrophoresis in 1.5% agarose gels.


Result

3.1 Identification and distribution of FOC in banana

The Komada medium was used to isolate F. oxysporum from banana plants. The results showed that Fusarium colonies were obtained only from symptomatic plants; none was found in the asymptomatic plants (Table1). Moreover, there were significant differences among the amounts of Fusarium spp. at different positions of diseased bananas. The fungi existed mostly at base of pseudostem with a concentration of 10.4 ×102 cfu/g, following with middle of pseudostem (4.76×102 cfu/g), root (1.70×102 cfu/g), top of pseudostem (0.98×102 cfu/g) and rhizome (0.24×102 cfu/g). Fusarium existed least in the rhizome, only 14% and 2% of those in the root and the base of pseudostem. All the isolates were identified as F. oxysporum by FOF1/FOR1 primer set with the amplification of a single 340-bp DNA fragment (Fig. 1), among which 40% of the isolates from root were not Foc4 identified by the Foc1 /Foc2 primer set ((Table1and Fig. 1).


Table 1 Distribution of Fusarium spp. inside asymptomatic and symptomatic banana plants

Sample

Type of plant

Sampled position

Fusarium colony

(×102 cfu/g)

Percentage of Fo (%)

Percentage of Foc (%)

AS-Ⅰ

Asymptomatic plant

Root

0

0

0

AS-Ⅱ

Rhizome

0

0

0

AS-Ⅲ

Base of pseudostem

0

0

0

AS-Ⅳ

Middle of pseudostem

0

0

0

AS-Ⅴ

Top of pseudostem

0




0

S-Ⅰ

Symptomatic plant

Root

1.70±0.10 c

100

60%

S-Ⅱ

Rhizome

0.24±0.08 c

100

100

S-Ⅲ

Base of pseudostem

10.4±1.73 a

100

100

S-Ⅳ

Middle of pseudostem

4.76±0.46 b

100

100

S-Ⅴ

Top of pseudostem

0.98±0.22 c

100

100




M

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22





Figure 1 Amplification of PCR products of 30 random samples which were from 150 samples of diseased plant using primer set FOF1/FOR1 (upper panel) and Foc-1 ⁄ Foc-2 (lower panel). Lane 1-4, partial samples from diseased roots(S-Ⅰ); 5-8, partial samples from diseased Rhizome (S-Ⅱ); 9-12, partial samples from diseased base of pseudostem (S-Ⅲ); 13-16, partial sampl es from diseased middle of pseudostem (S-Ⅳ); 17-20, partial sampl es from diseased top of pseudostem (S-Ⅴ); 21: positive control; 22: negative control using sterile dH2O as the template; M = molecular markers of Gen-100bp DNA ladder.


2.2 Transformation of FOC with gfp gene

2.2.1 Fungal strain, Media and vectors for transformation

Foc strain FJAT-3076, race 4, isolated in 2.1 were used for the GFP transformation. The fungus has been tested for its aggressiveness on banana cv. Cavendish, resulting in the wilting of 100% (thirty of thirty plants) of banana plants in up to 24 d. Strain FJAT-3076 was kept frozen in 20% glycerol at –80°C.

To optimize the transformation, the growth of FJAT-3076 was tested on PDA media with different concentrations of hygromicin B (Roche Diagnostics GmbH, Germany). The most efficient dose for selection of transformants was confirmed to be 150 mg L-1. The transformation vector (pCT74), which expressed sGFP from the ToxA promoter of Pyrenophora tritici-repentis, was obtained from LM ciuffetti (Freitag and Jacobs 1999; Lorang et al. 2001). Plasmids were propagated according to (Sambrook et al. 1989), and extracted using the Qiagen plasmid mini kit procedure (Qiagen, Cat 12123, USA).
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