Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4

  • Ying-Hong Lin
  • Jing-Yi Chang
  • En-Tzu Liu
  • Chih-Ping Chao
  • Jenn-Wen Huang
  • Pi-Fang Linda Chang
Article

Abstract

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.

Keywords

Molecular detection Panama disease PCR RAPD Reliable diagnosis 

Introduction

Fusarium wilt of banana (Musa spp.), commonly known as Panama disease caused by Fusarium oxysporum f. sp. cubense Snyder and Hanson (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; O’Donnell et al. 1998). Foc in infected soil can survive as a saprophyte for numerous years, and start to infect banana pseudostems during plant cultivation (Beckman and Roberts 1995; O’Donnell et al. 1998). The disruption of water translocation in vascular tissues leads to typical wilt symptoms including foliage chlorosis, necrosis and ultimately drooping of the leaves (Beckman 1990).

Based on the specific pathogenicity toward banana cultivars, Foc can be divided into three races (Groenewald et al. 2006). Race 1 is pathogenic to ‘Gros Michel (genome type = AAA)’ and ‘Silk (AAB)’ (Stover and Malo 1972; Su et al. 1986), while race 2 infects ‘Bluggoe’ (AAB) and other closely related cooking bananas (Moore et al. 1995; Waite and Stover 1960). Race 4 mainly causes disease in Cavendish cultivars as well as those susceptible to race 1 and race 2 (Hwang and Ko 2004; Su et al. 1977). Race 4 is further divided into tropical (T4) and subtropical (S4). The Foc S4 attacks Cavendish cultivars in subtropical areas such as Taiwan, Canary Islands, South Africa, and Australia (Brake et al. 1990; Gerlach et al. 2000; Ploetz 1990; Ploetz et al. 1990; Su et al. 1986). Conversely, the Foc T4 invades Cavendish cultivars in the tropical regions of Southeast Asia and Australia (Bentley et al. 1998; Ploetz 1994; Ploetz and Pegg 2000). To date no Foc T4 has been reported in Taiwan; however, in the recent survey in 2004, some Taiwan isolates of Foc were grouped as VCG01213/16 (Linda J Smith, personal communication) which was defined as T4 (tropical race 4) according to Bentley et al. (1998) and Ploetz and Pegg (2000).

To date, few effective and economically safe methods to protect banana from Fusarium wilt disease have been developed (Forsyth et al. 2006). The best way to manage Fusarium wilt disease at the present time is based on plant breeding for resistant lines (Diener and Ausubel 2005). It appears that a rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. Conventional field identification of the Panama disease pathogen is time-consuming and destructive (Daniells et al. 1995). Identification of Foc is usually based on morphological characteristics, which requires a great knowledge of Fusarium taxonomy (Jurado et al. 2006). Furthermore, Fusarium identification with microscopy is inefficient and difficult to detect in plant tissues, which may contain various disease complexes (Yergeau et al. 2005). A quick diagnostic assay for Foc would reduce the use of infected-symptomless plant materials for planting and propagation in plant breeding for resistant lines. Therefore, it is of vital importance that a sensitive and specific diagnostic method to identify Foc pathogen is available.

DNA fingerprinting with random amplification of polymorphic DNA (RAPD) is one of the powerful molecular tools for fungal pathogen identifications (Fungaro et al. 2004; Koike et al. 1995; Mes et al. 1999). The nucleotide amplification with polymerase chain reaction (PCR) is effective and specific, and its high sensitivity allows a direct identification of the pathogen in complex mixtures even when fungal mycelia are invisible under the microscope (Jurado et al. 2006). In this study, we have developed a novel primer set Foc-1/Foc-2 for specific molecular identification and detection of Foc race 4 isolates in Taiwan. Preliminary results have been presented (Chang et al. 2003).

Materials and methods

Growth of fungal and bacterial species

Fusarium wilt pathogens including 96 Foc race 4 isolates in Taiwan confirmed by pathogenicity tests on banana cv. Cavendish, seven reference Foc isolates (ATCC76247 and ATCC96285, race 1; ATCC76257 and ATCC96288, race 2; ATCC76262, ATCC96289, and ATCC96290, subtropical race 4), nine other F. oxysporum formae speciales, and one Fusarium verticillioides were used in this study (Table 1). The genomic DNA (gDNA) from two other fungal pathogens (Colletotrichum and Phytophthora) and one bacterium (Xanthomonas) were used for comparison (Table 1). A single-spore culture of each tested Fusarium isolate was grown on a Nash-PCNB plate (1.5% peptone, 2% agar, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.1% pentachloronitrobenzene, 0.03% streptomycin, and 0.1% neomycin; Nash and Snyder 1962). Colletotrichum gloeosporioides and Phytophthora infestans were grown on potato dextrose agar (PDA) plates (200 g l−1 of potato extracts, 1% glucose, and 2% agar). Single colonies of Xanthomonas oryzae pv. oryzae were grown on peptone sucrose agar (PSA) plate (1% peptone, 1% sucrose, 0.1% glutamic acid, pH 7.0, and 2% agar).
Table 1

Isolates of Fusarium and other fungal and bacterial pathogens used for PCR amplification

Species

Races

Original hosts

Geographic locations

Providers

F. oxysporum f. sp. cubense

 

Banana (Musa sp.)

  

ATCC76247 (VCG 0126)

1

Banana

Honduras

ATCC

ATCC96285 (VCG 0124)

1

Banana

SE. Queensland, Australia

ATCC

ATCC76257 (VCG 0124a)

2

Banana

Honduras

ATCC

ATCC96288 (VCG 0128)

2

Banana

N. Queensland, Australia

ATCC

ATCC76262 (VCG 0121)

S4

Banana

Taiwan

ATCC

ATCC96289 (VCG 0120)

S4

Banana

SE. Queensland, Australia

ATCC

ATCC96290 (VCG 0129)

S4

Banana

SE. Queensland, Australia

ATCC

Foc-7-9

4

Banana

Chiyi, Taiwan

TBRI

Foc-6-2

4

Banana

Hualien, Taiwan

TBRI

Foc-6-3

4

Banana

Hualien, Taiwan

TBRI

Foc-6-5

4

Banana

Hualien, Taiwan

TBRI

Foc-6-6

4

Banana

Hualien, Taiwan

TBRI

Foc-4-1

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-2 (VCG 01213/16b)

T4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-4

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-5

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-6

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-7

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-8

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-10

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-4-11

4

Banana

Kaohsiung, Taiwan

TBRI

Foc-TN3

4

Banana

Kaohsiung, Taiwan

ARI

Foc-TN5

4

Banana

Kaohsiung, Taiwan

ARI

Foc-7-13

4

Banana

Nantow, Taiwan

TBRI

Foc-7-14

4

Banana

Nantow, Taiwan

TBRI

Foc-7-16

4

Banana

Nantow, Taiwan

TBRI

Foc-7-17

4

Banana

Nantow, Taiwan

TBRI

Foc-7-18

4

Banana

Nantow, Taiwan

TBRI

Foc-7-19

4

Banana

Nantow, Taiwan

TBRI

Foc-7-20

4

Banana

Nantow, Taiwan

TBRI

Foc-7-21

4

Banana

Nantow, Taiwan

TBRI

Foc-7-22

4

Banana

Nantow, Taiwan

TBRI

Foc-7-23

4

Banana

Nantow, Taiwan

TBRI

Foc-T105

4

Banana

Nantow, Taiwan

J.-W. Huang

Foc-T202

4

Banana

Nantow, Taiwan

J.-W. Huang

Foc-3-1

4

Banana

Pingtung, Taiwan

TBRI

Foc-3-3

4

Banana

Pingtung, Taiwan

TBRI

Foc-3-15

4

Banana

Pingtung, Taiwan

TBRI

Foc-3-19

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-1

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-3

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-5

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-7

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-13

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-21

4

Banana

Pingtung, Taiwan

TBRI

Foc-5-25

4

Banana

Pingtung, Taiwan

TBRI

Foc-6-7

4

Banana

Taitung, Taiwan

TBRI

Foc-6-8

4

Banana

Taitung, Taiwan

TBRI

Foc-6-10

4

Banana

Taitung, Taiwan

TBRI

Foc-6-11

4

Banana

Taitung, Taiwan

TBRI

Foc-6-12

4

Banana

Taitung, Taiwan

TBRI

Foc-6-13

4

Banana

Taitung, Taiwan

TBRI

Foc-6-14

4

Banana

Taitung, Taiwan

TBRI

Foc-6-15

4

Banana

Taitung, Taiwan

TBRI

Foc-6-16

4

Banana

Taitung, Taiwan

TBRI

Foc-6-17

4

Banana

Taitung, Taiwan

TBRI

Foc-6-18

4

Banana

Taitung, Taiwan

TBRI

Foc-6-19

4

Banana

Taitung, Taiwan

TBRI

Foc-6-20

4

Banana

Taitung, Taiwan

TBRI

Foc-6-21

4

Banana

Taitung, Taiwan

TBRI

Foc-6-22

4

Banana

Taitung, Taiwan

TBRI

Foc-6-23

4

Banana

Taitung, Taiwan

TBRI

Foc-6-24

4

Banana

Taitung, Taiwan

TBRI

Foc-6-25

4

Banana

Taitung, Taiwan

TBRI

Foc-6-26

4

Banana

Taitung, Taiwan

TBRI

Foc-7-1

4

Banana

Tainan, Taiwan

TBRI

Foc-7-3

4

Banana

Taitung, Taiwan

TBRI

Foc-7-4

4

Banana

Taitung, Taiwan

TBRI

Foc-7-5

4

Banana

Taitung, Taiwan

TBRI

Foc-7-6

4

Banana

Taitung, Taiwan

TBRI

Foc-7-7

4

Banana

Taitung, Taiwan

TBRI

Foc-7-8

4

Banana

Taitung, Taiwan

TBRI

Foc-T12

4

Banana

Taitung, Taiwan

TDARES

Foc-T13

4

Banana

Taitung, Taiwan

TDARES

Foc-T14

4

Banana

Taitung, Taiwan

TDARES

Foc-T31

4

Banana

Taitung, Taiwan

TDARES

Foc-T33

4

Banana

Taitung, Taiwan

TDARES

Foc-T34

4

Banana

Taitung, Taiwan

TDARES

Foc-T35

4

Banana

Taitung, Taiwan

TDARES

Foc-T36

4

Banana

Taitung, Taiwan

TDARES

Foc-T37

4

Banana

Taitung, Taiwan

TDARES

Foc-T38

4

Banana

Taitung, Taiwan

TDARES

Foc-T41

4

Banana

Taitung, Taiwan

TDARES

Foc-T43

4

Banana

Taitung, Taiwan

TDARES

Foc-T44

4

Banana

Taitung, Taiwan

TDARES

Foc-1

4

Banana

Taiwan

TBRI

Foc-3

4

Banana

Taiwan

TBRI

Foc-5

4

Banana

Taiwan

TBRI

Foc-7

4

Banana

Taiwan

TBRI

Foc-8

4

Banana

Taiwan

TBRI

Foc-9

4

Banana

Taiwan

TBRI

Foc-14

4

Banana

Taiwan

TBRI

Foc-15

4

Banana

Taiwan

TBRI

Foc-21

4

Banana

Taiwan

TBRI

Foc-22

4

Banana

Taiwan

TBRI

Foc-23

4

Banana

Taiwan

TBRI

Foc-24

4

Banana

Taiwan

TBRI

Foc-25

4

Banana

Taiwan

TBRI

Foc-26

4

Banana

Taiwan

TBRI

Foc-27

4

Banana

Taiwan

TBRI

Foc-28

4

Banana

Taiwan

TBRI

Foc-29

4

Banana

Taiwan

TBRI

Foc-30

4

Banana

Taiwan

TBRI

F. oxysporum f. sp. chrysanthemi

Foch-11-28

 

Garland chrysanthemum (C. coronarium)

Changhua, Taiwan

J.-W. Huang

F. oxysporum f. sp. Lactucum

Fola-11-13

 

Lettuce (L. sativa)

Yunlin, Taiwan

J.-W. Huang

Fola-32-14

 

Lettuce

Yunlin, Taiwan

J.-W. Huang

F. oxysporum f. sp. lilii

Foli-F16

 

Lily (Lilium Oriental hybrid ‘Casa Blanca’)

Changhua, Taiwan

J.-W. Huang

Foli-G16

 

Lily

Changhua, Taiwan

J.-W. Huang

F. oxysporum f. sp. Luffae

Folu-S167

 

Loofah (L. cylindrica)

Nantow, Taiwan

Y.-S. Lin

Folu-114

 

Loofah

Nantow, Taiwan

Y.-S. Lin

F. oxysporum f. sp. lycopersici

Foly-4

 

Tomato (S. lycopersicum)

WVC/AVRDC

Foly-8

 

Tomato

WVC/AVRDC

F. oxysporum f. sp. Momordicae

Fom-101

 

Balsam pear (M. charantia)

Changhua, Taiwan

Y.-S. Lin

F. oxysporum f. sp. Niveum

Fon-H0103

 

Watermelon (C. lanatus)

Miaoli, Taiwan

J.-W. Huang

F. oxysporum f. sp. raphani

For-4566

 

Radish (R. sativus)

Nantow, Taiwan

J.-W. Huang

F. oxysporum

Fo-F66

 

Anoectochilus (A. formosanus)

Nantow, Taiwan

S.-P.-Y. Hsieh

F. verticillioides

Fv

 

Rice (O. sativa)

Tantiz, Taiwan

J.-W. Huang

C. gloeosporioides

Coll

 

Banana

Taichung, Taiwan

P.-F. L. Chang

P. infestans

Phyi109

 

Tomato

Changhua, Taiwan

J.-W. Huang

X. oryzae pv. Oryzae

Xoo

 

Rice

ARI

S4 Subtropical race 4, T4 tropical race 4, ATCC American-Type Culture Collection (Manassas, VA, USA), TBRI Taiwan Banana Research Institute (Pingtung, Taiwan), WVC/AVRDC The World Vegetable Centre (AVRDC, Tainan, Taiwan), ARI Agricultural Research Institute (Taichung, Taiwan), VCG vegetative compatibility group

aVCG of ATCC76257 was according to Ploetz and Correll (1988)

bVCG of Foc-4-2 was identified by Miss Linda J. Smith from the Plant Protection Unit, Queensland Department of Primary Industries, QDPI, Australia.

DNA isolation

Genomic DNA was extracted according to Dellaporta et al. (1983) with minor modifications. Dried fungal mycelium (1 g), overnight-grown bacterial culture (1.5 ml), and banana pseudostem tissues were snap frozen in liquid nitrogen and ground to fine powders using a mortar and pestle. DNA was extracted with 5 ml modified TNE buffer (100 mM Tris-HCl, pH 8.0; 50 mM Na2EDTA, pH 8.0; 50 mM NaCl; 8 μM β-mercaptoethanol; 1% SDS and 10 μg ml−1 RNase) and incubated at 65°C for 30 min. A 0.33× volume of 5 M potassium acetate was added, mixed and centrifuged at 20,000× g for 5 min. The supernatant was transferred into a fresh tube, and mixed with an equal volume of isopropanol to precipitate crude DNA. The samples were incubated at −20°C for 20 min and centrifuged at 4°C, 20,000× g for 20 min. The DNA pellet was resuspended in 200 μl dH2O, and an equal volume of chloroform/isoamyl alcohol (24:1; v/v) was added and mixed thoroughly. After centrifugation at 4°C, 20,000× g for 5 min, the upper aqueous phase was transferred to a fresh tube, and 0.1× volume of 3 M sodium acetate (pH 5.2) and 2.5× volume of absolute ethanol were added. After centrifugation at 4°C, 20,000× g for 5 min, the supernatant was decanted and the DNA pellet was washed with 300 μl 75% ethanol, allowed to air dry, and finally dissolved in 1× TE buffer (10 mM Tris–HCl, pH 8; 0.1 mM EDTA) for further analysis.

Primer design and RAPD analysis

In order to obtain nucleotide markers that could specifically differentiate Foc race 4 from Foc races 1 and 2, F. oxysporum formae speciales infecting other plants and Fusarium species, more than 100 random decamers were used for PCR amplification. For RAPD analysis, a 25 μl PCR mixture contained 50 ng gDNA, 1× reaction buffer (10 mM Tris–HCl, pH9.0; 50 mM KC1; 2.5 mM MgCl2), 0.1 mM of each dNTP, 0.8 μM random decamer primer (Operon Technologies Inc., Alameda, CA, USA), and 2.5 unit Taq DNA polymerase (MDBio, Inc., Taipei, Taiwan). The parameters for PCR were denatured at 94°C for 90 s, followed by 30 cycles of denaturing at 94°C for 30 s, touchdown annealing temperatures (30 s) at 36°C, 34°C, and 32°C for 5, 5, and 20 cycles, respectively, and polymerising at 72°C for 60 s, and then a final extension at 72°C for 10 min. RAPD products were subjected to electrophoresis in 2.0% agarose gels.

Specific PCR and sensitivity experiment of the assay

For the sensitivity experiment, a 50 μl PCR mixture contained 200 to 10−5 ng Foc-24 gDNA, 1× reaction buffer (10 mM Tris–HCl, pH 9.0; 50 mM KC1; 2.5 mM MgCl2), 0.1 mM of each dNTP, 0.4 μM of each specific primer, and 2.5 unit Taq DNA polymerase (MDBio, Inc., Taipei, Taiwan). 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.

Southern hybridisation

DNA gel blots of RAPD and PCR products were subjected to Southern hybridisation using the Foc race 4-specific DNA fragment of RAPD as a probe. After gel electrophoresis, DNA was transferred to nylon membranes (GeneScreen Hybridisation Transfer Membrane, PerkinElmer Life Sciences, Inc., Boston, MA, USA). Probe randomly biotinylated labelling, DNA hybridisation (at 68°C), and subsequent chemiluminescent detection were carried out using NEBlot® Phototope® Kit and Phototope®-Star Detection Kit (New England BioLabs, Inc., Ipswich, MA, USA) following the instruction manuals.

Results

Screening of a RAPD marker specific to F. oxysporum f. sp. cubense race 4

A RAPD fragment specific to Foc race 4 isolates from Taiwan was amplified by the random primer OP-A02, 5′-TGCCGAGCTG (Fig. 1). This PCR fragment (called OPA02404) was purified, cloned into pGEM®-T Easy vector (Promega Co, Madison, WI, USA), and sequenced. The nucleotide sequence (accession number: EU379562) and Southern hybridisation data confirmed that this RAPD fragment was 404 bp long and had the original primer sequence (OP-A02) at both ends. This sequence has been published by Liu (2003) in our group, and it matched the sequence of a F. oxysporum f. sp. cubense isolate FOC-FT marker for molecular detection and quantitation genomic sequence (accession number: EF155535) deposited to GenBank in December of 2006.
Fig. 1

Fingerprinting of random amplified polymorphic DNA and their Southern blot hybridisation in Fusarium oxysporum species. Genomic DNA of 11 F. oxysporum f. sp. cubense race 4 isolates, 13 isolates of other F. oxysporum formae speciales, and one F. verticillioides (Fv), one Colletotrichum (Coll), one Phytophthora (Phyi109), one bacterium (Xanthomonas oryzae pv. oryzae, Xoo), and the disease-free banana pseudostem (Banana-C) were used as templates (see Table 1) for amplification by using random primer OP-A02. The RAPD products were subjected to Southern blot hybridisation (shown as the panel below each ethidium bromide-stained DNA gel pattern with light background) using the Biotin-labelled OP-A02 amplified fragment OPA02404 as a probe. The 404-bp size DNA band specific to Foc race 4 is indicated on the left. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder (GeneMark Technology Co., Ltd., Tainan, Taiwan)

Specificity and the PCR amplification

In order to develop a molecular detection system for Foc race 4 isolates in Taiwan, a specific primer set Foc-1/Foc-2 (5′-CAGGGGATGTATGAGGAGGCT/5′-GTGACAGCGTCGTCTAGTTCC) was designed from the OPA02404 nucleotide sequence (accession number EU379562, nt79-nt99/nt300-nt320) for PCR amplification. A 242-bp size fragment was produced by PCR from Foc race 4 gDNA. This primer set was able to amplify the corresponding DNA fragment of 242 bp (called Foc242) only from gDNA of 11 Foc race 4 isolates in Taiwan but not from gDNA of any other tested isolates (Fig. 2). The specificity is the most important premise for developing a molecular diagnosis protocol. Therefore, we confirmed that the Foc242 marker is present in all 99 tested isolates of Foc race 4 (96 Taiwanese and three reference isolates as listed in Table 1; see supplement data for the other Taiwanese isolates not shown in Figs. 2 and 3). Moreover, using the gDNA of seven Foc reference isolates (race 1, 2, and S4) from the American-Type Culture Collection and one T4 isolate (Foc-4-2), both PCR products, OPA02404 (Fig. 3a) and Foc242 (Fig. 3b), were present in all the tested Foc race 4 isolates (both T4 and S4) but not in the tested race 1 and 2 isolates.
Fig. 2

Amplification of PCR products using the primer set Foc-1/Foc-2 specific to Fusarium oxysporum f. sp. cubense race 4 isolates. The fungal isolates, bacterial pathogen, and plant material used for extracting genomic DNA as PCR templates (50 ng) are listed in Fig. 1 and Table 1. The location of a 242-bp DNA band specific to F. oxysporum f. sp. cubense race 4 isolates (labelled as Foc) is indicated on the left. Banana-C = disease-free banana pseudostem gDNA as the PCR template. M = molecular markers of Gen-100 DNA ladder

Fig. 3

Ethidium bromide-stained and DNA gel blot hybridisation patterns of (a) RAPDs and (b) PCR products using genomic DNA samples of Fusarium oxysporum f. sp. cubense subtropical (S4) and tropical (T4) race 4 isolates as templates. The primers used for RAPDs and PCR were random primer OP-A02 and the specific primer set Foc-1/Foc-2, respectively. All tested gDNA samples of the seven reference isolates (labelled as ‘ATCC isolates’) and four Foc race 4 isolates from Taiwan (labelled as ‘Foc race 4’), including the Foc-4-2 isolate which belongs to T4, are as listed in Table 1. The DNA gels were subjected to Southern hybridisation (shown as the panel below each ethidium bromide-stained DNA gel pattern with light background) using the Biotin-labelled OPA02404 as a probe. The locations of 404- and 242-bp DNA bands specific to Foc race 4 isolates are indicated on the left. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder

Sensitivity of the Foc race 4-specific PCR

Serial dilutions of gDNA of Foc-24 isolate, ranging from 200 to 10−5 ng, were prepared to test the sensitivity of Foc race 4-specific PCR assay. It appeared that primer set Foc-1/Foc-2 was able to amplify the 242 bp-size fragment as low as 10 pg (10−2 ng) of gDNA in a 25 μl reaction mixture (Fig. 4a). Using OPA02404 fragment (which includes the whole region of Foc242) as a probe for Southern hybridisation, the sensitivity to detect Foc242 PCR product was enhanced to 0.1 pg (10−4 ng; Fig. 4b). In addition, the sensitivity of PCR assays with primer set Foc-1/Foc-2 was not affected with different amounts of banana gDNA (50 ng to 2 μg) added to the reaction mixture containing 25 ng of Foc-24 gDNA (Fig. 5).
Fig. 4

Detection sensitivity of the primer set Foc-1/Foc-2 amplified fragment in genomic DNA of Fusarium oxysporum f. sp. cubense race 4 isolate Foc-24. A serial of dilutions of Foc-24 gDNA ranging from 200 to 10–5 ng were used as templates. The PCR products (a) were subjected to Southern blot hybridisation (b) using the Biotin-labelled amplified fragment OPA02404 as a probe. The location of the 242-bp size DNA band specific to Foc race 4 is indicated on the right. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder

Fig. 5

Effect of plant genomic DNA on the detection sensitivity of the primer set Foc-1/Foc-2 amplified DNA fragment in Fusarium oxysporum f. sp. cubense race 4 isolate Foc-24. In 50 ng gDNA of Foc-24 isolate, 0.05 to 2 μg gDNA of disease-free banana pseudostem was added to each PCR. The location of a 242-bp DNA band specific to Foc race 4 is indicated on the left. 0 = positive control using only 50 ng Foc-24 gDNA without any banana gDNA as the PCR template. B = PCR control using 2 μg gDNA of disease-free banana pseudostem only as the template. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder

In order to determine the sensitivity of our PCR detection system using primer set Foc-1/Foc-2 against fungal mycelia present in banana tissues, gDNAs were extracted from the mixtures of dried Foc-24 mycelia and fresh diseased-free banana pseudostem at various ratios. Fifty nanogram of gDNA mixture was subjected to PCR amplification. The results indicated that as low a ratio of 0.005 (dry weight of fungal mycelia to fresh weight of banana pseudostem, 1 g of fresh weight equals to 0.06 g of dry weight of banana pseudostem) could generate the Foc242 DNA fragment by PCR (Fig. 6).
Fig. 6

Detection sensitivity of the primer set Foc-1/Foc-2 amplified fragment in dried mycelia of Fusarium oxysporum f. sp. cubense race 4 isolate Foc-24 in the presence of disease-free banana tissues. Genomic DNA was extracted from dried fungal mycelia plus fresh plant tissues with a different ratio (0.005 to 50) of fungal dry weight to plant fresh weight. For each PCR, 50 ng of total gDNA was used as the template. The location of a 242-bp DNA band specific to Foc race 4 is indicated on the left. B = PCR control using 1 μg gDNA of disease-free banana pseudostem only as the template. N = negative control using sterile dH2O as the PCR template. P = positive control using 50 ng Foc-24 gDNA as the PCR template. M = molecular markers of Gen-100 DNA ladder

Foc-1/Foc-2, the primer set specific to Foc race 4 isolates, was also used to test naturally infected banana tissues collected from the field. It appeared that the Foc242 DNA fragment could easily be amplified in gDNA of symptomatic banana pseudostems, collected from two different fields, by PCR (Fig. 7, lanes 1–6). In addition, in the gDNA of symptomless banana leaves collected from the same two fields, Foc242 DNA was also amplified even though the amplicons were very faint (Fig. 7 lanes 7–12).
Fig. 7

Molecular detection of Fusarium oxysporum f. sp. cubense race 4 in naturally infected banana tissues. Genomic DNA samples extracted from two symptomatic pseudostems (A and B) and two symptomless leaves (C and D) of bananas collected from two different fields were used for PCR by primer set Foc-1/Foc-2. Three regions (10 cm2 each) of each pseudostem (lanes 13 for A, and lanes 46 for B) and leaf (lanes 79 for C, and lanes 1012 for D) were sampled for gDNA extraction for PCR amplification. For each PCR, 50 ng of total gDNA was used as the template. The location of a 242-bp DNA band specific to Foc race 4 is indicated on the left. BS = PCR control using 50 ng gDNA of disease-free tissue-cultured banana pseudostem as the template. BL = PCR control using 50 ng gDNA of disease-free tissue-cultured banana leaf as the template. N = negative control using sterile dH2O as the PCR template. P = positive control using 50 ng Foc-24 gDNA as the PCR template. M = molecular markers of Gen-100 DNA ladder

Discussion

PCR assays have been implemented successfully for identification and detection of economically important Fusarium species such as Fusarium avenaceum (Schilling et al. 1996; Turner et al. 1998), Fusarium culmorum (Klemsdal and Elen 2006; Nicholson et al. 1998; Schilling et al. 1996), Fusarium graminearum (Nicholson et al. 1998; Schilling et al. 1996; Yoder and Christianson 1998), Fusarium langsethiae (Wilson et al. 2004), Fusarium moniliforme (the official name is now F. verticillioides; Möller et al. 1999), Fusarium subglutinans (Möller et al. 1999), Fusarium poae (Parry and Nicholson 1996), Fusarium sambucinum (Yoder and Christianson 1998), Fusarium sporotrichioides (Wilson et al. 2004) and Fusarium venenatum (Yoder and Christianson 1998). Most of these molecular techniques are based on the development of species-specific primers.

The OPA02404 DNA marker was not amplified from gDNA of nine tested F. oxysporum formae speciales, which were not banana pathogens, one non-pathogenic F. oxysporum isolated from banana field soil (Fo-DK1; Chang 2005), one other Fusarium sp., two other fungal species (C. gloeosporioides and P. infestans), and one bacterium strain (X. oryzae pv. oryzae). Furthermore, different race isolates from two different geographic regions (ATCC76247 and ATCC96285 for race 1; ATCC76257 and ATCC96288 for race 2; ATCC76262, ATCC96289, and ATCC96290 for subtropical race 4) showed dissimilar RAPD patterns with the random primer OP-A02 amplification. Moreover, the OP-A02 RAPD patterns of the three subtropical race 4 (S4) isolates tested were similar but different from that of the tropical race 4 (T4) isolate (Foc-4-2). In addition, Foc242 was present in all Foc race 4 (both S4 and T4) isolates tested including 96 Taiwanese and three reference S4 (ATCC76262, ATCC96289, and ATCC96290) isolates. Therefore, the OPA02404 and Foc242 amplified fragments of Foc isolates could be used as molecular markers for identification and detection of Foc race 4 in Taiwan. In the future, more Foc isolates from various geographic origins, races differentiated by pathogenicity tests, will be used to confirm the specificity of our PCR assay method to the tropical race 4 (T4) isolates worldwide.

The Foc-1/Foc-2 primer set occasionally amplified an unexpected amplicon of approximately 300 bp in our PCR assays. This DNA fragment, named Foc303 according to Chang (2005), was sequenced and compared with that of the Foc242 (Chang 2005). This Foc303 fragment was only amplified by the Foc-2 primer. However, the DNA sequences of Foc242 and Foc303 are dissimilar.

The set of Foc-1/Foc-2 primers presented in this work allowed us to process small amounts of DNA samples and obtain the detection results within hours, in comparison with the time-consuming traditional agar plating or pathogenicity tests which may take days or weeks. Agar plating to differentiate Fusarium species requires the knowledge of morphological characters whereas pathogenicity tests to identify formae speciales of F. oxysporum are labour-intensive.

The sensitivity of our PCR assay was comparable to those reported for F. culmorum, one of the causal agents of wheat head blight disease (Nicholson et al. 1998; Schilling et al. 1996). However, with a nested PCR method, the detection sensitivity was increased up to 5–50 fg for the detection of F. culmorum in cereal samples (Klemsdal and Elen 2006). For PCR, usage of more than one primer pair in a reaction could result in a higher probability of cross-annealing with non-selective templates, such as plant gDNA, to generate non-specific PCR products, which might interfere with the result interpretation. In our study, amplification of the Foc race 4-specific marker, Foc242, was not affected by the presence of banana gDNA. Therefore Foc242 is suitable for detecting pathogen Foc in infected banana.

It appears that our PCR diagnosis protocol with the primer set Foc-1/Foc-2 was applicable to screen naturally Foc-infected banana samples. Even though positive results were obtained by PCR, however, we were able to recover Foc only from the diseased banana pseudostems but not the symptomless banana leaves by a plate-out assay. These results suggest that the PCR assay we developed here is more sensitive than the plate-out assay. Nevertheless, we could not rule out the possibility that the leaf samples (0.5 × 2 cm2 of leaf area) for the plate-out assay were free of Foc, but the nearby leaf area (at least 10 times larger than those for the plate-out assay) picked for PCR contained Foc. On the other hand, in combination with Southern blot hybridisation, our PCR assay with the Foc-1/Foc-2 primer set could increase sensitivity 100-fold. Therefore, the developed molecular detection method with the primer set Foc-1/Foc-2 may lead to efficient disease management practices for banana production due to quick and accurate diagnosis of Fusarium wilt disease, be beneficial to rapidly test the presence of Foc race 4 in breeding materials of banana for resistance to Foc race 4, and be useful for basic research in epidemiology and fungal population genetics (Schilling et al. 1996).

Notes

Acknowledgements

We thank Dr. Peter P. Ueng of USDA-ARS for providing DNA samples of reference isolates of Foc race 1, 2, and 4, and his critical review of this manuscript. We are grateful to Drs. Y.-S. Lin (NCHU), K.-S. Chen (FTHEB, ARI), S.-P.-Y. Hsieh (NCHU), S.-C. Hwang (TBRI), Miss H.-L. Lee (TDARES), WVC/AVRDC, and ARI for providing the tested microorganisms, and to Dr. W.-H. Ko for critical reading and useful suggestions for this manuscript. We also thank Miss L.J. Smith for VCG identification, Dr. R.C. Ploetz for information about Foc race 4, Miss Y.-L. Wan and Mr. C.-C. Su for technical assistance. This research was supported in part by Bureau of Animal and Plant Health Inspection and Quarantine, and Department of International Affairs, Council of Agriculture, Executive Yuan, Taiwan, R.O.C. under grant numbers 89ST-6.2-BQ-65(06), 91AS-7.3.1-BQ-B2(3), 93AS-1.9.2-BQ-B1, 96AS-4.1.2-IC-I1(2), and 97AS-4.1.2-IC-I1(2); by the Ministry of Education, Taiwan, R.O.C. under the ATU plan; and also by National Chung Hsing University, Taiwan, R.O.C.

Supplementary material

10658_2008_9372_MOESM1_ESM.doc (3.3 mb)
ESM 1Amplification of PCR products using the primer set Foc-1/Foc-2 specific to Fusarium oxysporum f. sp. cubense (Foc) race 4 isolates. The fungal isolates used for extracting genomic DNA (gDNA) as PCR templates (50 ng) were as listed in Table 1. The DNA gels were subjected to Southern hybridisation (shown as the panel below each ethidium bromide-stained DNA gel pattern with light background) using the Biotin-labelled OPA02404 as a probe. The location of a 242-bp DNA band specific to F. oxysporum f. sp. cubense race 4 isolates (labeled as Foc) is indicated on the left. Fon-H0103 is the Fusarium wilt pathogen of watermelon to serve as a negative control here. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder (DOC 3.34 MB).

References

  1. Beckman, C. H. (1990). Host responses to the pathogen. In R. C. Ploetz (Ed.), Fusarium wilt of banana (pp. 107–114). St. Paul: APS.Google Scholar
  2. Beckman, C. H., & Roberts, E. M. (1995). On the nature and genetic basis for resistance and tolerance to fungal wilt diseases of plants. Advances in Botanical Research, 21, 35–77. doi:10.1016/S0065-2296(08)60008-7.CrossRefGoogle Scholar
  3. Bentley, S., Pegg, K. G., Moore, N. Y., Davis, R. D., & Buddenhagen, I. W. (1998). Genetic variation among vegetative compatibility groups of Fusarium oxysporum f. sp. Cubense analysed by DNA fingerprinting. Phytopathology, 88, 1283–1288. doi:10.1094/PHYTO.1998.88.12.1283.PubMedCrossRefGoogle Scholar
  4. Brake, V. M., Pegg, K. G., Irwin, J. A. G., & Langdon, P. W. (1990). Vegetative compatibility groups within Australian populations of Fusarium oxysporum f. sp. cubense, the cause of Fusarium wilt of bananas. Australian Journal of Agricultural Research, 41, 863–870. doi:10.1071/AR9900863.CrossRefGoogle Scholar
  5. Chang, J. Y. (2005). Molecular identification of Fusarium oxysporum f. sp. cubense and its detection in infected banana seedlings. Taichung, Taiwan, ROC: National Chung Hsing University, Master’s thesis.Google Scholar
  6. Chang, P. F. L., Chang, C. Y., Lin, E. T., Chen, I. R., & Huang, J. W. (2003). Detection of Fusarium oxysporum f. sp. cubense based on RAPD and PCR analysis. Plant Pathology Bulletin, 12, 277 Abstract in Chinese.Google Scholar
  7. Daniells, J., Davis, D., Peterson, R., & Pegg, K. (1995). Goldfinger: Not as resistant to sigatoka/yellow sigatoka as first thought. Infomusa, 4, 6.Google Scholar
  8. Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA mini-preparation: version II. Plant Molecular Biology Reporter, 1, 19–21. doi:10.1007/BF02712670.CrossRefGoogle Scholar
  9. Diener, A. C., & Ausubel, F. M. (2005). Resistance to Fusarium oxysporum 1, a dominant Arabidopsis disease-resistance gene, is not race specific. Genetics, 171, 305–321. doi:10.1534/genetics.105.042218.PubMedCrossRefGoogle Scholar
  10. Forsyth, L. M., Smith, L. J., & Aitken, E. A. B. (2006). Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity. Mycological Research, 110, 929–935. doi:10.1016/j.mycres.2006.03.008.PubMedCrossRefGoogle Scholar
  11. Fungaro, M. H. P., Vissotto, P. C., Sartori, D., Vilas-Boas, L. A., Furlaneto, M. C., & Taniwaki, M. H. (2004). A molecular method for detection of Aspergillus carbonarius in coffee beans. Current Microbiology, 49, 123–127. doi:10.1007/s00284-004-4273-z.PubMedGoogle Scholar
  12. Gerlach, K. S., Bentley, S., Moore, N. Y., Pegg, K. G., & Aitken, A. B. (2000). Characterisation of Australian isolates of Fusarium oxysporum f. sp. cubense by DNA fingerprinting analysis. Australian Journal of Agricultural Research, 51, 945–953. doi:10.1071/AR99172.CrossRefGoogle Scholar
  13. Getha, K., & Vikineswary, S. (2002). Antagonistic effects of Streptomyces violaceusniger strain G10 on Fusarium oxysporum f. sp. cubense race 4: indirect evidence for the role of antibiosis in the antagonistic process. Journal of Industrial Microbiology & Biotechnology, 28, 303–310. doi:10.1038/sj.jim.7000247.CrossRefGoogle Scholar
  14. Groenewald, S., Van Den Berg, N., Marasas, W. F., & Viljoen, A. (2006). The application of high-throughput AFLPs in assessing genetic diversity in Fusarium oxysporum f. sp. cubense. Mycological Research, 110, 297–305. doi:10.1016/j.mycres.2005.10.004.PubMedCrossRefGoogle Scholar
  15. Hwang, S. C., & Ko, W. H. (2004). Cavendish banana cultivars resistant to Fusarium wilt acquired through somaclonal variation in Taiwan. Plant Disease, 88, 580–588. doi:10.1094/PDIS.2004.88.6.580.CrossRefGoogle Scholar
  16. Jurado, M., Vázquez, C., Marín, S., Sanchis, V., & González-Jaéna, M. T. (2006). PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize. Systematic and Applied Microbiology, 29, 681–689. doi:10.1016/j.syapm.2006.01.014.PubMedCrossRefGoogle Scholar
  17. Klemsdal, S. S., & Elen, O. (2006). Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples. Letters in Applied Microbiology, 42, 544–548. doi:10.1111/j.1472-765X.2006.01880.x.PubMedCrossRefGoogle Scholar
  18. Koike, M., Watanabe, M., Nagao, H., & Ohshima, S. (1995). Molecular analysis of Japanese isolates of Verticillium dahliae and V. alboatrum. Letters in Applied Microbiology, 21, 75–78. doi:10.1111/j.1472-765X.1995.tb01010.x.PubMedCrossRefGoogle Scholar
  19. Liu, E. T. (2003). Analysis of Fusarium oxysporum f. sp. cubense using random amplified polymorphic DNA and polymerase chain reaction techniques. Taichung, Taiwan, ROC: National Chung Hsing University, Master’s thesis.Google Scholar
  20. Mes, J. J., Weststeijn, E. A., Herlaar, F., Lambalk, J. J. M., Wijbrandi, J., Haring, M. A., et al. (1999). Biological and molecular characterization of Fusarium oxysporum f. sp. lycopersici divides race 1 isolates into separate virulence groups. Phytopathology, 89, 156–160. doi:10.1094/PHYTO.1999.89.2.156.PubMedCrossRefGoogle Scholar
  21. Möller, E. M., Chełkowski, J., & Geiger, H. H. (1999). Species-specific PCR assays for the fungal pathogens Fusarium moniliforme and Fusarium subglutinans and their application to diagnose maize ear rot disease. Journal of Phytopathology, 147, 497–508. doi:10.1111/j.1439-0434.1999.tb03856.x.CrossRefGoogle Scholar
  22. Moore, N. Y., Bentley, S., Pegg, K. G., & Jones, D. R.(1995). Fusarium wilt of banana. Musa Disease Fact Sheet no. 5, International Network for the Improvement of Banana and Plantain, Montpellier, France.Google Scholar
  23. Nash, S. M., & Snyder, W. C. (1962). Quantitative estimations by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopathology, 52, 567–572.Google Scholar
  24. Nicholson, P., Simpson, D. R., Weston, G., Rezanoor, H. N., Lees, A. K., Parry, D. W., et al. (1998). Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology, 53, 17–37. doi:10.1006/pmpp.1998.0170.CrossRefGoogle Scholar
  25. O’Donnell, K., Kistler, H. C., Cigelnik, E., & Ploetz, R. C. (1998). Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies. Proceedings of the National Academy of Sciences of the United States of America, 95, 2044–2049. doi:10.1073/pnas.95.5.2044.PubMedCrossRefGoogle Scholar
  26. Parry, D. W., & Nicholson, P. (1996). Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathology, 45, 383–391. doi:10.1046/j.1365-3059.1996.d01-133.x.CrossRefGoogle Scholar
  27. Ploetz, R. C. (1990). Population biology of Fusarium oxysporum f. sp. cubense. In R. C. Ploetz (Ed.), Fusarium wilt of banana (pp. 63–76). St. Paul: APS.Google Scholar
  28. Ploetz, R. C. (1994). Panama disease: Return of the first banana menace. International Journal of Pest Management, 40, 326–336.CrossRefGoogle Scholar
  29. Ploetz, R. C., & Correll, J. C. (1988). Vegetative compatibility among races of Fusarium oxysporum f. sp. cubense. Plant Disease, 72, 325–328. doi:10.1094/PD-72-0325.CrossRefGoogle Scholar
  30. Ploetz, R. C., Herbert, J., Sebasigari, K., Hernandez, J. H., Pegg, K. G., Ventura, J. A., et al. (1990). Importance of Fusarium wilt in different banana growing regions. In R. C. Ploetz (Ed.), Fusarium wilt of banana (pp. 9–26). St. Paul: APS.Google Scholar
  31. Ploetz, R. C., & Pegg, K. G. (2000). Fusarium wilt. In D. R. Jones (Ed.), Diseases of banana, abacá and enset (pp. 143–159). Wallingford: CABI.Google Scholar
  32. Schilling, A. G., Möller, E. M., & Geiger, H. H. (1996). Polymerase chain reaction-based assays for species-specific detection of Fusarium culmorum, F. graminearum, and F. avenaceum. Phytopathology, 86, 515–523. doi:10.1094/Phyto-86-515.CrossRefGoogle Scholar
  33. Snyder, W., & Hanson, H. (1940). The species concept in Fusarium. American Journal of Botany, 27, 64–67. doi:10.2307/2436688.CrossRefGoogle Scholar
  34. Stover, R. H., & Malo, S. E. (1972). The occurrence of fusarial wilt in normally resistance ‘dwarf Cavendish’ banana. Plant Disease Reporter, 56, 1000–1003.Google Scholar
  35. Su, H. J., Chuang, T. Y., & Kong, W. S. (1977). Physiological race of Fusarial wilt fungus attacking Cavendish banana of Taiwan. Special publication no. 2 pp. 1–21. Pingtung: Taiwan Banana Research Institute.Google Scholar
  36. Su, H. J., Hwang, S. C., & Ko, W. H. (1986). Fusarial wilt of Cavendish bananas in Taiwan. Plant Disease, 70, 814–818. doi:10.1094/PD-70-814.CrossRefGoogle Scholar
  37. Turner, A. S., Lees, A. K., Rezanoor, H. N., & Nicholson, P. (1998). Refinement of PCR-detection of Fusarium avenaceum and evidence from DNA marker studies for phenetic relatedness to Fusarium tricinctum. Plant Pathology, 47, 278–288. doi:10.1046/j.1365-3059.1998.00250.x.CrossRefGoogle Scholar
  38. Waite, B. H., & Stover, R. H. (1960). Studies on Fusarium wilt of bananas, VI. Variability and cultivar concept in Fusarium oxysporum f. sp. cubense. Canadian Journal of Botany, 38, 985–994. doi:10.1139/b60-087.CrossRefGoogle Scholar
  39. Wilson, A., Simpson, D., Chandler, E., Jennings, P., & Nicholson, P. (2004). Development of PCR assays for the detection and differentiation of Fusarium sporotrichioides and Fusarium langsethiae. FEMS Microbiology Letters, 233, 69–76. doi:10.1016/j.femsle.2004.01.040.PubMedCrossRefGoogle Scholar
  40. Yergeau, E., Filion, M., Vujanovic, V., & St-Arnaud, M. (2005). A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus. Journal of Microbiological Methods, 60, 143–154. doi:10.1016/j.mimet.2004.09.006.PubMedCrossRefGoogle Scholar
  41. Yoder, W. T., & Christianson, L. M. (1998). Species-specific primers resolve members of Fusarium section Fusarium taxonomic status of the edible “Quorn” fungus reevaluated. Fungal Genetics and Biology, 23, 68–80. doi:10.1006/fgbi.1997.1027.PubMedCrossRefGoogle Scholar

Copyright information

© KNPV 2008

Authors and Affiliations

  • Ying-Hong Lin
    • 1
  • Jing-Yi Chang
    • 1
  • En-Tzu Liu
    • 1
  • Chih-Ping Chao
    • 2
  • Jenn-Wen Huang
    • 1
  • Pi-Fang Linda Chang
    • 1
  1. 1.Department of Plant PathologyNational Chung Hsing UniversityTaichungRepublic of China
  2. 2.Taiwan Banana Research InstitutePingtungRepublic of China

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