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Eur J Plant Pathol (2016) 144:225–230 DOI 10.1007/s10658-015-0763-z

Evaluation of methods to detect the cotton wilt pathogen Fusarium oxysporum f. sp. vasinfectum race 4 Frankie K. Crutcher & Hung K. Doan & Alois A. Bell & R. Michael Davis & Robert D. Stipanovic & Robert L. Nichols & Jinggao Liu

Accepted: 9 September 2015 / Published online: 12 September 2015 # US Government 2015

Abstract Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is an economically important disease of cultivated cottons (Gossypium hirsutum and G. barbadense). Recently, Fov race 4 has spread among soils planted to cotton in the San Joaquin Valley of California causing significant losses. Because Fov 4 is seedborne and persists in infested soils, it is a potential threat to all US cotton production regions. Tests have been developed to rapidly identify race 4 in the laboratory and the field, including a race 4 specific PCR and a test kit for use with fungal mycelium and infected plant tissue. Currently, the test kit is the only available method to identify race 4 in the field. Both the PCR and kit tests were evaluated on a panel of 36 Fov isolates that represented all described races and many genotypes from several geographic regions. As expected, race 4 isolates gave positive reactions, as did race 7 isolates. One race 7 isolate was not vegetatively compatible with the race 4 isolates, while the other isolate of race 7 exhibited a compatible reaction. Isolates of race 7 and race 4 have identical partial F. K. Crutcher : A. A. Bell : R. D. Stipanovic : J. Liu (*) Southern Plains Agricultural Research Center, 2765 F&B Road, College Station, TX 77845, USA e-mail: [email protected] H. K. Doan : R. M. Davis Department of Plant Pathology, University of California, Davis, CA 95616, USA R. L. Nichols Cotton Incorporated, 6399 Weston Parkway, Cary, NC 27513, USA

sequences in three nuclear genes. In addition, the type isolate of race 3 gave a positive reaction. All other isolates, including the highly-virulent Australian isolates, were negative. The diagnostic tests clearly distinguished race 4 from races 1, 2, 6, and 8 but not from the race 3 and race 7 isolates tested here. Race 3 and 7 are similar in etiology to that of race 4, thus a method is needed to distinguish these in the field. Keywords Fusarium oxysporum . vasinfectum . Cotton . Race 4 . Detection

Fusarium wilt of cotton is caused by Fusarium oxysporum f. sp. vasinfectum (Fov) and occurs in most cotton growing regions of the world. Based on pathogenicity to cotton and non-cotton hosts, Fov has been described as comprising eight different races: races 1, 2, and 6 from the Americas (Armstrong and Armstrong 1978), race 7 and 8 from China (Chen et al. 1985), race 4 from India (Fahmy 1927; Armstrong and Armstrong 1960), race 3 from Egypt (Armstrong and Armstrong 1960), and race 5 from Sudan (Ibrahim 1966). Alternatively, the American races, 1, 2, and 6 have been grouped into a single race, designated A, using RAPD analysis (Assigbetse et al. 1994). Race 5 was retracted when it was shown to be identical to race 3 (Nirenberg et al. 1994). Race 8 is widespread in the United States (Holmes et al. 2009; Cianchetta et al. 2015; Bell, unpublished). A new biotype from Australia (Davis et al. 1996) and new molecular genotypes of Fov from the United States (Holmes et al. 2009) have also been

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described but not given race designations. In the San Joaquin Valley (SJV) of California, a particularly virulent form of race 4 was identified in 2001 (Kim et al. 2005). California race 4 can cause severe symptoms on cotton and shows no dependence on root-knot nematode (Meloidogyne incognita) for infection (Kim et al. 2005), whilst race 1 and 2 isolates generally caused only mild symptoms on Upland cotton varieties unless associated with the M. incognita (Garber et al. 1979). Control of Fov races 1 and 2 has often been achieved through controlling M. incognita, rather than the fungus itself (Hyer et al. 1979; Wang and Roberts 2006). California has been a major state for production of both cotton fiber and planting seed (Lawrence et al. 2015). Seed production in a Fov-infested site and dissemination through infested seed could be a mechanism for disease dispersal (Bennett et al. 2008). Currently, there are no effective seed treatments for Fov. Therefore, the development of rapid race 4 screening strategies is essential for preventing further spread of the disease beyond SJV of California. Two diagnostic methods have been developed to detect the fungus in soil and plants. One is a PCR method using race 4 specific primers developed from unique AFLP polymorphisms (Yang 2006). The other one is a kit based field diagnostic assay using the AmplifyRP® Acceler8™ kit. This kit was developed based on the same race 4 specific PCR primers as described in Yang (2006) with modifications. The kit utilizes two target specific primers with the reverse primer labelled with biotin and an internal probe labeled with fluorescein isothiocyanate (FITC) (Doan et al. 2014). It shows increased specificity compared to the PCR primers and is convenient for field identification. In this report, we describe the evaluation of the specificity of the Agdia AmplifyRP® Acceler8™ Diagnostic Assay Kit towards a panel of Fov isolates that represent distinct races, genotypes, and geographic origins. Fov isolates previously characterized by pathogenicity assays and partial sequencing of the elongation factor-1 alpha gene, phosphate permease gene, and βtubulin gene were maintained on potato dextrose agar (PDA) at 27 °C (Table 1). Vegetative compatibility analysis of the isolates was conducted according to Liu et al. (2011). For the kit test, 30 mg of mycelium was scraped off from a 10-day-old PDA culture inoculated with the specific fungal isolate and was ground in the sample mesh bag containing extraction buffer provided with the kit. To obtain mycelia for DNA extraction,

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potato dextrose broth (PDB, 50 ml) was inoculated with 1×106 conidia obtained from 4-day-old single-spored isolate cultures on PDA. PDB cultures were incubated at 25 °C for 3 days with shaking at 150 rpm (Innova®44, New Brunswick Scientific, Edison, NJ) and filtered through miracloth (Calbiochem®, EMD Millipore, Billerica, MA). The resulting mycelium was frozen in liquid nitrogen and maintained at −80 °C until DNA was extracted. Genomic DNA from Fov was extracted for both PCR analyses and the kit using a standard phenolchloroform extraction (Xu et al. 1996). The primers R4f 5′ GCTCCGTGTCWGAGCTTCTT 3′ and R4r 5′ GTTATGCTCCACGATGAGCA 3′ (Yang et al. 2006) were used to amplify the expected 208 bp amplicon from 50 ng of genomic DNA. Thermocycler conditions (10 cycles of 30 s each at 94 °C, 59 °C, and 72 °C, followed by 25 cycles of 30 s at 90 °C, 30 s at 59 °C, and 15 s at 72 °C) were used for a 20 μl PCR reaction using GoTaq® (Promega). The entire amplified product was loaded onto an agarose gel for electrophoresis. In addition, 5 μl aliquots of 20 μl PCR reactions with ITS primers (White et al. 1990), were used as loading controls. Specificity of the race 4 diagnostic kit was evaluated per manufacturer’s instructions. Ten μl of PD1 pellet diluent was added into a reaction pellet and vortexed vigorously. Using a pipettor, 1 μl of 50 ng/μl genomic DNA or 1 μl of homogenate from the kit sample mesh bag prepared from fungal tissue was added to the rehydrated pellet and mixed. The reaction was incubated in the 39 °C preheated block. After 15 min, the reaction tube was inserted in the reaction apparatus, which then was added to the detection chamber housing before closing. Results were interpreted after 20 min. The experiment was replicated twice for tissue and once for genomic DNA for each of the tested isolates. Experiments for both PCR and kit tests for a subset of isolates (6, 16, 203, 1072, 1143, 1144, 1763, 1814, 1817, 1857, and 1858) were independently repeated in the lab of R. M. Davis, University of California, Davis, CA. For any tested isolate, identical results were obtained for the PCR and the two kit tests either using DNA or fungal tissue samples. Most results obtained for nonrace 4 isolates were negative (Table 1 and Fig. 1). This included six Australian isolates, four of which produce prodigious amounts of fusaric acid and infect cotton seedlings similar to race 4 isolates from California (Liu et al. 2011). Although specificity of the PCR protocol

Eur J Plant Pathol (2016) 144:225–230

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Table 1 Specificity test of the race 4 specific PCR primers and the Agdia diagnostic kit on a panel of Fusarium oxysporum f. sp. vasinfectum isolates representing races, VCGs, and molecular lineages from several geographic regions Race, biotype, or lineagea

VCGb

Reference

Race 4 PCR

Kitc

SC

R1 (type isolate)

VCG0111

Fernandez et al. 1994

-

-

India

R4 (type isolate)

VCG0114

Fernandez et al. 1994

+

+ -

Isolate

Original name

Origin

6

ATCC 16421

9

ATCC 16613

11

ATCC 46644

CA

R1

VCG1A

Liu et al. 2011

-

16

ATCC 16611

SC

R2 (type isolate)

VCG0112

Fernandez et al. 1994

-

-

203

CDR203

SC

R8

VCG2*

-

-

218

CDR218

NC

R1

VCG1B*

-

-

233

CDR233

LA

R8

VCG2*

-

-

260

AK2-8a

AR

R8

VCG2*

-

-

538

NT1

TX

r3 lineage

Self*

-

-

930

CDR930

TX

R1

VCG1C*

-

-

932

CDR932

TX

R2

VCG0112*

-

-

943

CDR943

AL

r8 lineage

VCG10*

-

-

960

CDR960

AL

r6 lineage

VCG9*

-

-

1072

CDR1072

AL

r3 lineage

VCG13*

-

-

1089

Auk24232

Australia

Aus Biotype

VCG01111

Liu et al. 2011

-

-

1090

Auk24288

Australia

Aus Biotype

VCG01111

Liu et al. 2011

-

-

1091

Auk24299

Australia

Aus Biotype

VCG01111

Liu et al. 2011

-

-

1092

Auk24301

Australia

Aus Biotype

VCG01111

Liu et al. 2011

-

-

1141

AL-KL1

AL

r4 lineage

Self*

Bennett et al. 2011

-

-

1143

AL-KL11

AL

r4 lineage

Self*

Bennett et al. 2013

-

-

1144

MS-GL10

MS

r4 lineage

Self*

Bennett et al. 2013

-

-

1441

AuSeed5

Australia

r3 lineage

Aus2

Liu et al. 2011

-

-

1447

AuSeed14

Australia

r3 lineage

VCG01111

Liu et al. 2011

-

-

1760

NRRL 25433

China

R7

Self*

Skovgaard et al. 2001

+

+

1761

KL 10-101

AL

R8

VCG2*

-

-

1763

KL 10-308

AL

R8

VCG2*

-

-

1807

CA9

CA

R4

VCG0114*

+

+

1808

CA12

CA

R4

VCG0114*

Kim et al. 2005

+

+

1814

CA1

CA

R8

VCG2*

Kim et al. 2005

-

-

Kim et al. 2005

1815

CA3

CA

r3 lineage

Unk*

Kim et al. 2005

-

-

1817

CA11

CA

r3 lineage

Unk*

Kim et al. 2005

-

-

1818

CA14

CA

R4

VCG0114*

Kim et al. 2005

+

+

1819

CAS9

CA

R4

VCG0114*

Kim et al. 2005

+

+

1857

ATCC 16612

Egypt

R3 (type isolate)

VCG0113

Fernandez et al. 1994

+

+

1858

NRRL 31665

China

R8

VCG2*

Skovgaard et al. 2001

-

-

1861

NRRL 31672

China

R7

VCG0114*

Skovgaard et al. 2001

+

+

a Lineage designations were based on isolates’ sequence homology (partial sequences of elongation factor-1 alpha gene, phosphate permease gene, and β-tubulin gene) to those of corresponding race isolates. They only relate to molecular genotype, not the pathogenicity of the isolates of the referred race b

VCG: Vegetative Compatibility Group; VCG designations were either from this study denoted by asterisk (*) or from the cited reference; BSelf^ indicates that the isolate complements only with itself

c

Tests were performed using either the extracted DNA or mycelium tissue. Both tests gave identical results for any given isolate; therefore, the results given in the column represent both of the kit tests

228

Eur J Plant Pathol (2016) 144:225–230

Fig. 1 Results of the race 4 specific (top panel) and ITS control (bottom panel) PCR reactions. Numbered headings refer to isolate names from Table 1

has been evaluated with the Australian isolates (Yang 2006), this is the first test of specificity of the race 4 kit with the Australian isolates. Isolates that tested positive for the primers and kit were isolates 9 (India race 4 type isolate), 1760, 1861 (China race 7 isolates), 1807, 1808, 1818, 1819 (California race 4 isolates), and 1857 (race 3 type isolate) (Figs. 1 and 2). The tests were able to detect all of the race 4 isolates as expected. Race 7 is considered to be of the same lineage as race 4 and previous DNA sequencing was unable to distinguish the two races (Skovgaard et al. 2001). The race 7 isolate 1861

(NRRL 31672) is vegetatively compatible with race 4 type isolate, while race 7 isolate 1760 (NRRL 25433) failed to complement race 4 type isolate (Table 1). Regardless, both race 7 isolates tested positive by the primers and kit. Isolates 1141, 1143, and 1144 are only weakly pathogenic to cotton and have been described as Brace 4-like^ based on sequence homology of the elongation factor-1 alpha gene (Bennett et al. 2013); these isolates tested negative with PCR and the kit (Figs. 1 and 2). The race 3 type isolate ATCC 16612 (isolate 1857) has been noted to give a positive reaction in the race 4 specific PCR protocol (Yang 2006). This positive

Fig. 2 Test kit results of a California race 4 isolate 1807, a race 4 lineage isolate 1143, a race 7 isolate 1760, a California race 3 lineage isolate 1817, and the race 3 type isolate 1857 using fungal tissue. Lineages were based on isolates’ sequence homology (partial sequences of elongation factor-1 alpha gene, phosphate

permease gene, and β-tubulin gene) to those of corresponding race isolates. They only relate to molecular genotype, not the pathogenicity of the isolates of the referred race. Arrow 1 indicates the control line indicating proper functioning of the detection system. Arrow 2 indicates the race 4 specific testing line

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reaction was eliminated by increasing the annealing temperature of the PCR reaction from 59 °C to 67 °C. However, no amplicon was produced from any of the isolates at 67 °C in the present study (data not shown). The race 3 type isolate was not used initially to test the specificity of the race 4 kit (Doan et al. 2014), but it was included in this evaluation. The tests of the race 3 type isolate resulted in a strong positive reaction for both fungal tissue and genomic DNA (Fig. 2). Other isolates with sequences similar to race 3 (isolates 538, 1441, 1447, 1815, and 1817) did not give positive reactions with either the primers or the kit (Figs. 1 and 2). In conclusion, both published methods for identification of race 4 isolates are effective for the isolates tested here with the exception of the race 3 type isolate and the race 7 isolates. According to Skovgaard et al. (2001), race 7 is indistinguishable from race 4 in a phylogenetic study based on DNA sequences. Furthermore, some members of the two races were found to be vegetatively compatible (Table 1). This is the first report of positive reactions for the Fov race 4 Agdia AmplifyRP® Acceler8™ Diagnostic Assay Kit with race 3 and race 7. To date, isolates with DNA sequences identical to those of race 3 type isolate have not been detected in the US. Acknowledgments We thank Cotton Incorporated for their generous support of this research and Dr. Kathy S. Lawrence, Auburn University, for providing fungal isolates with KL designation.

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