Approaches for eliminating PCR inhibitors and designing PCR primers for the detection of phytopathogenic fungi

ARTICLE IN PRESS

Crop Protection 26 (2007) 145–161 www.elsevier.com/locate/cropro

Approaches for eliminating PCR inhibitors and designing PCR primers for the detection of phytopathogenic fungi Zhonghua Maa,, Themis J. Michailidesb, a

Institute of Biotechnology, Zhejiang University, Hangzhou 310029, PR China Department of Plant Pathology, University of California, Davis, Kearney Agricultural Center, 9240 S. Riverbend Avenue, Parlier, CA 93648, USA

b

Received 20 January 2006; received in revised form 5 April 2006; accepted 10 April 2006

Abstract The polymerase chain reaction (PCR) technology has been used to rapidly detect, characterize, and identify a variety of organisms. In a PCR diagnosis study, the development of PCR primers is one of the most important steps. This paper reviews several major approaches that have been used successfully for designing PCR primers specific to various phytopathogenic fungi. These approaches include using species-specific genes or DNA regions, or anonymous unique DNA regions to design PCR primers. Since the problem with PCR inhibitors is prevalent in PCR diagnosis of plant diseases, we also review various techniques that have been used to circumvent PCR inhibitors derived from plant tissues, soil, air, and water samples. r 2006 Elsevier Ltd. All rights reserved. Keywords: PCR diagnostics; PCR inhibitors; Phytopathogenic fungi

1. Introduction The polymerase chain reaction (PCR) technique has revolutionized molecular biology since it was first described in 1985 (Saiki et al., 1985). After the introduction of thermostable Taq DNA polymerase from Thermus aquaticus and the development of automated oligonucleotide synthesis and thermocyclers, PCR-based techniques have been used in various biological studies ranging from the identification of novel genes and pathogens to the quantification of characterized nucleotide sequences, and provided insights into the intricacies of single cells as well as the evolution of species. Pathogen and disease diagnoses are fundamental to virtually all aspects that relate to plant pathology. Accurate identification and early detection of plant pathogens is the cornerstone of successful disease management. Because many pathogens are difficult to identify based only on Also to be corresponded to. Corresponding author. Tel.: +1559 646 6546; fax: +1559 646 6593.

E-mail addresses: [email protected] (Z. Ma), [email protected] (T.J. Michailides). 0261-2194/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2006.04.014

morphological characteristics, and morphological identification of plant pathogens is also time consuming, affected by environmental conditions, and requires extensive knowledge in taxonomy, PCR-based assays have become powerful tools for the rapid diagnosis of plant diseases although the role of the traditional assay continues to be an important method in plant pathology. Normally, conventional PCR amplification involves repeated cycles of template denaturation, primer annealing, and polymerase extension to amplify a specific sequence of DNA determined by two oligonucleotide primers. Obviously, the development of PCR primers specific to target organisms is one of the most important steps in PCR diagnostics. The ability of an oligonucleotide to serve as a PCR primer may depend on the kinetics of association and dissociation of primer–template duplexes at the annealing and extension temperatures, and on duplex stability of mismatched nucleotides and their location. The primers which are unique for the target sequence to be amplified should fulfill certain criteria such as primer length, GC%, annealing and melting temperature, 50 end stability, 30 end specificity, etc. (Dieffenbach et al., 1993). This paper reviews multiple methods for screening unique DNA sequences used for the design of PCR

ARTICLE IN PRESS 146

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

primers. Since the quantitative real-time PCR has been one of the most promising methodologies for detection and characterization nucleotide sequences, this paper also reviews the use of real-time PCR for detection of phytopathogenic fungi. Additionally, in this paper, we also summarized various methods used to circumvent PCR inhibitors in plant, soil, air, and water samples since the prevalence of inhibitors in PCR diagnosis of plant diseases is a major problem and has been less discussed in other literature reviews. It is beyond the scope of this review to discuss advantages and limitations of molecular diagnostics, PCRbased techniques, including conventional PCR, multiplex PCR, quantitative PCR, and, BIO–PCR (a combined biological and enzymatic amplification), magnetic-hybridization-PCR, and immunocapture-PCR, which have been presented and well discussed in several other literature reviews (Louws et al., 1999; Martin et al., 2000; Schaad & Frederick, 2002; McCartney et al., 2003; Schaad et al., 2003). 2. Methods for PCR primer design 2.1. Use of species-specific genes or DNA regions for design of PCR primers 2.1.1. Ribosomal DNA Design of PCR primers using sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) has gained widespread usage in PCR diagnosis of phytopathogenic fungi. To date, PCR primers designed based on ITS sequences have been used to detect more than 80 pathogenic fungal species, and many of them can be found from the on-line primer searchable database at http://www.sppadbase.com. The genes of subunits of rDNA (18S, 5.8S, and 28S) of fungi have evolved slowly and are useful for studying distantly related taxonomic groups at genera or above levels. Whereas, the two ITS regions and intergenic spacer (IGS) of the nuclear rDNA evolve fast and are frequently used for differentiation of species (White et al., 1990). Since rDNA is highly repeated in fungi, primers developed based on rDNA could be highly sensitive. To design species-specific PCR primers, sequences of target fungal rDNA could be obtained from some databases (e.g. http://www.ncbi.nlm.nih.gov, http://rrna.uia.ac.be/ primers/database.html), or by amplifying the target rDNA fragment with conserved primers (White et al., 1990). After comparing the sequences of the target rDNA with those of related fungal species by alignment with a computer program (e.g. Clustal W at http://www.ebi.ac.uk/clustalw), the sequences specific to the target fungal pathogen could be found, and subsequently, species-specific PCR primers could be developed using computer software (e.g. primer3 at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). For instance, to develop a PCR assay for diagnosis of panicle and shoot blight of pistachio in California caused by the pycnidial stage (a Fusicoccum sp.) of the ascomycete

Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not., the ITS regions of Fusicoccum sp. were amplified and sequenced from a number of isolates collected from different locations in California. When compared the complete sequences of the ITS regions from Fusicoccum sp. with those from other related fungal species, unique sequences in ITS regions of Fusicoccum sp. from pistachio were found. A pair of primers (BDI and BDII) specifically targeting Fusicoccum sp. rDNA was designed based on the unique sequences. This pair of primers amplified a 356-bp DNA fragment from each of 300 Fusicoccum sp. isolates collected from pistachio and other hosts throughout California in different years, but not for other 33 fungal species isolated from pistachio in California and isolates of Fusicoccum sp. obtained from pistachio trees in Greece. DNA fingerprints also showed that the isolates of Fusicoccum sp. collected from California pistachio were genetically different from those collected from Greece (Ma & Michailides, 2002). The PCR assay using this primer pair can be used to rapidly (within a few hours) identify Fusicoccum sp. on pistachio in California, whereas, identification of this pathogen based on morphological characteristics requires at least 2 weeks until pycnidia with mature pycnidiospores are formed. 2.1.2. Allele-specific PCR Since ITS regions are highly conserved among some genetically related fungi (such as Monilinia fructicola, M. laxa, and M. fructigena), there may be only few base (even one) pair differences among ITS sequences of these species. In such circumstances, allele-specific PCR primers designed based on single base-pair change could be used to detect these related fungal species. Because the terminals of primers are of vital importance for a successful amplification and the 30 -end position in the primer affects PCR amplification more dramatically than mismatches at other positions. In an allele-specific PCR assay, one or both PCR primers are designed to match the desired bases of target sequence and mismatch others in non-target species at the 30 end of primers. This technique has been used successfully to develop a PCR method for the identification of genetically related species M. laxa and M. fructicola by Ioos & Frey (2000). In this study, based on the different base-pair at nucleotide position 108 in the ITS sequences of M. fructicola and M. laxa (Fig. 1(A)), the authors designed the forward primers ITS1Mfcl and ITS1Mlx, which were specific to M. fructicola and M. laxa, respectively, and the different base between these two primers was located at the 30 -ends (Fig. 1(B)). Similarly, based on the different base pair difference at position 422 in the ITS sequences of these two species, two reverse primers were designed (Fig. 1(B)). Using these two pairs of allele-specific primers, these two fungal species can be differentiated easily. 2.1.3. Other conserved genes than rDNA Species-specific PCR primers could also be designed based on other conserved genes (e.g. the b-tubulin, the

ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

M. fructicola M. laxa

147

86 96 106 416 426 436 …TGTATGCTCG CCAGAGGATA ATTAAACTCT……GTTCTC AGTG TGCTTCTGCC AAAACCCAAA… …TGTATGCTCG CCAGAGAATA ATCAAACTCT……GTTCTC GGTG TGCTTCTGCC AAAACCCA AA…

(A) Primer sequences (5’-3’) for: M. fructicola

ITS1Mfcl TATGCT CGCCAGAGGATAATT

ITS4Mfcl TGGCTTTTGGCAGAAGCACACT

M. laxa

ITS1Mlx

ITS4Ml x

TATGCTCGCCAGAGAATAATC

TGGGTTTTGGCAGAAGCACACC

(B) Fig. 1. (A) Sequence alignment of the ITS1 and ITS2 regions showing species-specific base differences (in bold italic) between Monilinia fructicola and M. laxa. The regions chosen for the design of the species-specific primers are underlined. (B) The respective forward and reverse species-specific primers for detection of M. fructicola and M. laxa.

elongation factor, and ascomycete mating-type genes) (Table 1) when rDNA sequence variation or conservation is not suitable for developing primers for a target pathogen. Such genes may have little variation in their translated amino acid sequences, but their third codon position and intron regions appear to have relatively high rates of nucleotide substitutions. In addition to the rDNA, the btubulin gene perhaps the most common target for the development of PCR primers. Similar to amplification of rDNA regions, b-tubulin gene from a target fungus could be amplified by using degenerate primers (McKay et al., 1998). After sequencing and comparing the sequences of the target b-tubulin gene to those of related fungal species, the unique sequences specific to the target fungal pathogen could be found for developing species-specific PCR primes. For instance, in a study on diagnosis of M. laxa, one of two causal agents (M. laxa and M. fructicola) of brown rot of stone fruit and almond in California, we found that the deduced amino acid sequence of the b-tubulin gene from M. laxa were more than 99% identical to that from a closely related species, M. fructicola, but the partial sequence of the intron No. 6 of b-tubulin genes from these two pathogens had up to 40% dissimilarity. Based on the sequence of intron No. 6, we developed a pair of primers, which can clearly differentiate M. laxa from other closely related species in a few hours (Ma et al.,2005). 2.1.4. Genes involved in mycotoxigenesis Food and feed contamination by mycotoxins is of great concern because many mycotoxins are carcinogenic and they are not easily removed during food processing. Although toxin abundance does not correlate with fungal contamination, it is linked to the toxigenic properties of each microbial strain. PCR primers designed based on genes involved in mycotoxigenesis can be used to detect toxigenic strains, subsequently, real-time PCR assays can be developed for quantitative detection of toxigenic fungi in food. Negative results indicate that a sample should be virtually free of mycotoxins, only the positive samples need to be analyzed for the presence of mycotoxins using physico-chemical standard methods. For instance, a realtime PCR assay using primers designed based on the nor-1 gene coding the norsolorinic acid reductase, one of the first genes in the aflatoxin biosynthetic pathway, was developed

to detect aflatoxigenic Aspergillus flavus in plant-type foods like maize, pepper, and paprika (Mayer et al., 2003). Additionally, detection of groups of species producing toxins, rather than specific species, has also been conducted by using primers developed from sequences of toxin cluster genes. Ward et al. (2002) developed chemotype-specific PCR primers based on Tri3 and Tri12 genes for the detection of various chemotype Fusarium graminearum (Ward et al., 2002), the fungal pathogen that contaminates wheat with deoxynivalenol (DON). 2.1.5. Mitochondrial DNA Fungal mitochondrial DNA (mtDNA) has been used widely as a source of molecular markers for genetic diversity and taxonomic studies. The relative small size of the mtDNA having conserved and variable regions makes it suitable for assessment of genetic variation and differentiation of closely related species (Mills, 1994). Because of its small size with high copy number and the existence of conserved and variable regions, cloning mtDNA may be less time-consuming for identification of species-specific sequences than screening of random clones of genomic DNA (Martin, 1991). Several studies showed that the identification of variable regions of the mitochondrial genome and selection of unique sequences provided an effective approach for the development of PCR primers specific to Alternaria, Tilletia, Pythium, and Phytophthora spp. (Table 1). Sequences with high A+T content (490%) are often refractory to amplification. Considering the extraordinary high A+T content of mitochondrial DNA, fragments larger than 1.5-kb are almost impossible to be amplified under standard reaction conditions (Rondan-Duenas et al., 1999). Thus, specific PCR conditions are required for amplification of long mitochondrial DNA sequences. Rondan-Duenas et al. (1999) reported an increase in the relative concentration of dATP and dTTP (0.33 mM dATP, dTTP each, and 0.10 mM dGTP, dCTP each) greatly improves the amplification of such A+T-rich DNA sequences. Su et al. (1996) found that reduction of the PCR extension temperature from 72 to 60 1C allows amplification of this refractory A+T-rich DNA. Results from these studies suggested that a low extension temperature and high concentration of dATP and dTTP versus dGTP and

Primer name: sequence (50 –30 )

AF: ACACTGCTTCAGCATTTTTCTTCATAG AR: TTGTCCAATTCATGGTATAGCACTCA

BT5: GCTCTAGACTGCTTTCTGGCAGACC BTAFR: YATGCTTGCACTCCCTTCGC

BT5: GCTCTAGACTGCTTTCTGGCAGACC BTFUS: TTTTGCATGCATTCCTTGCC

BT5: GCTCTAGACTGCTTTCTGGCAGACC BTPAS: TTGCCGGATGCCTGTTGGGG

BT5: GCTCTAGACTGCTTTCTGGCAGACC BTPUR: TCGCACAGTTTAGCATGACC

BT5: GCTCTAGACTGCTTTCTGGCAGACC BTSOR: CATCCATCTGCCCAACGATT

Tox5-1: GCTGCTCATCACTTTGCTCAG. Tox5-2: CTGATCTGGTCACGCTCATC

MAT1p2: AGAAACTGACTGATACATCAAGGGG MAT1p3: TCATAAGAAGTGTTGAAGGAATCACAG

GcHMG1: CTTTACCGTAAGGAGCGTCACCAT GcHMG2: TGATCCGCCATCTGCTTGTAGAGT

GaoA-V2: AGGGACAATAAGTGCAGA GaoA-R2: ACTGTGCACTGTCGCAAGTG

Fgtubf: GGTCTCGACAGCAATGGTGTT Fgtubr:GCTTGTGTTTTTCGTGGCAGT FGtubTM probe: (FAMACAACGGAACGGCACCTCTGAGCTCCAGC-TAMRA)

FOA28: ATCCCCGTAAAGCCCTGAAGC TL3: GGTCGTCCGCAGAGTATACCGGC

For race 2 R2.1: CTTGTTTCTCGATTTCTGTCTCACG Ft3: GGCGATCTTGATTGTATTGTGGTG For race 1 or 8 R8.1: CGATGAAGTCGGTTTGCGATT Ft3: GGCGATCTTGATTGTATTGTGGTG For race 4 IMP1: GCGGATCGGTTATGACGG R4.2: GGTGATTGGAGGAGGAATACC

Fsg1: GTCTTCTAGGATGGGCTGGT Fsg2: CATTTAATGCCTAGTCCCCTATCA

FsgEF1: GAGTCGGTTAGCTTCTGTC FsgEF2: GCGCGCCTTGCTATTCTCC

Effp-1: AACCCCGCCCGAGGACTCA Effp-2: AGACATGAGCGATGAGAGGCA

Fungal species

Alternaria spp.

Claviceps africana

Claviceps fusiformis

Claviceps paspali

Claviceps purpurea

Claviceps sorghicola

Fusarium spp.

F. circinatum MAT-1

F. circinatum MAT-2

F. graminearum

F. graminearum

F. oxysporum f.sp. albedinis

F. oxysporum f. sp. dianthi

F. solani f. sp. glycines

F. solani.f. sp . glycines

F. solani f. sp. phaseoli

1315

438

237

Impalad

mtSSUe EF-1af

562

395

Fot1

EF-1a

564

400

Fot1c

Fot1

111

898

GaoAb

b-tubulin gene

187

Mating-type gene

380

658

Tri5a

Mating-type gene

190

b-tubulin gene

227

234

209

209

226

Product size (bp)

72

Touchdown PCR

Touchdown PCR

Touchdown PCR

62

67

56

70

70

63

60

60

60

60

60

68

Annealing temperature (1C)

Filion et al. (2003)

Li & Hartman (2003)

Li & Hartman (2003)

Chioccetti et al. (1999)

Fernandez et al. (1998)

Reischer et al. (2004)

Niessen and Vogel (1997)

Schweigkofler et al. (2004)

Schweigkofler et al. (2004)

Niessen, and Vogel (1998)

Tooley et al. (2001)

Tooley et al. (2001)

Tooley et al. (2001)

Tooley et al. (2001)

Tooley et al. (2001)

Ma et al. (2003a)

Reference

148

b-tubulin gene

b-tubulin gene

b-tubulin gene

b-tubulin gene

Cytochrome b gene

Target DNA

Table 1 PCR primers developed based on sequences of specific genes for the detection of phytopathogenic fungi

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

Gfmat2c: AGCGTCATTATTCGATCAAG GFmat2d: CTACGTTGAGAGCTGTACAG

pfh2a: CGTCACACGTTCTTCAACC pfh2b: CGTTTCACGCTTCTCCG

MLF2: CGAGGCTCTTTACGACATTTG MLR2: TTATACTATGGCCGGGCAGA

E1: CGGTATGGGAACACTTCTCATCAG STSP2R: GTAACGACCGTTGCGGAAATCGCT

Mnem-1:AATAAAATTAATTTTAATATATAATTAG FMnem-3: dTATGTTTAATATCTGTAAATAATAG

PNF: CCACCACGCAGCAAACTGCGGC PNR: TTGAGTACCAGGCCGCTCGTAG

IL7: CTCCAGCTAGTCAAGCCTAG IL8: CACCGTCAACTCGCAGTC

FMPps1: AGTTTCATTAGAAGATTATTTAC FMPps2: AAAATTGTTTGATTTTATTAAGTATC PpsCALOrange probe CAL Orange d(TTAATAAAAAAATTATGATATTTAAACTAATTGGT) BHQ-1

FMPr-1: GTATTTAAAATCATAGGTGTAATTTG FMPr-7: TGGTTTTTTTAATTTATATTATCAATG Probe: 6-FAM d(CAGATATTAAACAAATTATATATAAAATCAAACAA) BHQ-1

Nested-PCR External primer pair, PBAW-10: CCCCGGGGATCACGATAAATAACA PBAW-11: GGAAGGCCGCCCAGGACTACC

G. fujikuroi, MAT-2

Magnaporthe oryzae

Monilinia laxa

Mycosphaerella graminicola

Phytophthora nemorosa

P. nicotianae

P. nicotianae

P. pseudosyringae

P. ramorum

Plasmodiophora brassicae

BR3: TCCCAAAGCAAGCCCAAATACACG BR2: GAATGTTTCACAGCAGCTGCTGGT

YRNT1: CTTCAAGATCGGTGGCCTGACCGA YRNT2: GTGAGCTGTGAAGGGATCGCGGGA

Pb1: CAACATTGCCTGGTATTGAGAAAC Pb2: ATCTGATACGCCTACACCGTCC

P1: GTTCGTTTGTTTGGGGATACG RP2: CTTCGTACTTAACCAACCAGC

BAF4ST: GACCAATTCGGCACCCTCAGTGTA SNSP7: CCGGTCAGCTCAACTCTGACCTGA

STIF2: ACTCACAATCCTCATTCGACGCGA BAF4ST: GACCAATTCGGCACCCTCAGTGTA

MT5315: AGGAGGGCTACTGGAGGTG MT3311: GTAATTGGACCCACGAGACAAG

Puccinia recondita

Puccinia striiformis

Pyrenopeziza brassicae

Pythium aphanidermatum

Septoria nodorum

S. tritici

Tapesia acuformis

Internal primer pair, PBTZS-3: CCACGTCGATCACGTTGCAAT PBTZS-4: CCTGGCGTTGATGTACTGGAA

GFmat1a: GTTCATCAAAGGGCAAGCG GFmat1b: TAAGCGCCCTCTTAACGCCTTC

Gibberella fujikuroi, MAT-1

Mating-type gene

b-tubullin gene

b-tubulin gene

mtDNA region

Mating-type gene

b-tubullin gene

b-tubullin gene

Isopentyltransferase gene

mtDNA

mtDNA

Elicitin gene parA1

Elicitin gene parA1

mtDNA

b-tubulin gene

b-tubulin gene

Pot2 transposon

Mating-type gene

Mating-type gene

812- and 418-bp

555 and/or 561

464

650

750

351

300

774 398

134

158

378

239

100

496

356

687

800

200

55

65

65

68

68

65

65

72 72

55

55

65

65

64

65

65

55

67

67

Dyer et al. (2001)

Fraaije et al. (2001)

Fraaije et al. (2001)

Wang et al. (2002)

Foster et al. (1999)

Fraaije et al. (2001)

Fraaije et al. (2001)

Ito et al. (1999); Wallenhammar & Arwidsson (2001)

Tooley et al. (2006)

Tooley et al. (2006)

Lacourt & Duncan (1997)

Kong et al. (2003)

Martin et al. (2004)

Fraaije et al. (1999)

Ma et al. (2005)

Harmon et al. (2003)

Steenkamp et al. (2000)

Steenkamp et al. (2000)

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161 149

TI17M1: TCCCCTTGGATCAGAACGTA TI17M2: AGAAGTCTAACTCCCCCCTCT

T. indica

Real-time PCR Tin11: TAATGTTGGCGTGGCGGCAT Tin10: AGCTCCGCCTCAAGTTCCTC TaqMan probe: ATTCCCGGCTTCGGCGTCACT

b

Tri5: the trichodiene synthase gene. GaoA: the galactose oxidase gene. c Fot1: the transposable element Fot1. d Impala: the transposable element impala. e mtSSU: the mitochondrial small-subunit ribosomal DNA. f EF-1a: the translation elongation factor 1-a gene.

a

T. walkeri

mtDNA region

212

212

391

Tin11: TAATGTTGGCGTGGCGGCAT Tin10; AGCTCCGCCTCAAGTTCCTC

mtDNA region

2300

118

825

212

414

Tin7: GTTTGAGCCACGCTATGACC Tin8: GGCTCATCTACGCATACGTT

T. walkeri

mtDNA region

mtDNA region

mtDNA region

Tin11: TAATGTTGGCGTGGCGGCAT Tin4: CAACTCCAGTGATGGCTCCG

Ti-1: TGGGCTGAGTCTGAGATGC Ti-4: AGTAATACCTGCGTCTCATAGC

T. indica

TI57M1: TTTTCCCTCTCTCCTTTTTTCA TI57M2: AGCAAAGACAAAGTAGGCTTCC

Real-time PCR Tin3: CAATGTTGGCGTGGCGGCGC Tin10: AGCTCCGCCTCAAGTTCCTC TaqMan probe: ATTCCCGGCTTCGGCGTCACT

414

Tin3: CAATGTTGGCGTGGCGGCGC Tin4: CAACTCCAGTGATGGCTCCG

60

55

65

65

65

60

60

60

65

65

65

65

65

55

Annealing temperature (1C)

Frederick et al. (2000)

Frederick et al. (2000)

Ferreira et al. (1996)

Smith et al. (1996)

Frederick et al. (2000)

Frederick et al. (2000)

Dyer et al. (2001)

Reference

150

T. indica

392

Tin5: GACGTCGAGGCCGACCGTAT Tin6: GGCGGACTACCACTCGAGCT

212

885

mtDNA region

812- and 418-bp

Tin3: CAATGTTGGCGTGGCGGCGC Tin6: GGCGGACTACCACTCGAGCT

Tin3: CAATGTTGGCGTGGCGGCGC Tin10: AGCTCCGCCTCAAGTTCCTC

Tilletia indica

Mating-type genes (MAT-1 and MAT-2)

Product size (bp)

497

MT5315: AGGAGGGCTACTGGAGGTG MT317: GTTACAGCGATGACTCCAGCG

T. yallundae

Target DNA

F3: GGCACCAGAGTACAGCTGTCGTT3 R1: GTCGGATTTGCGGACACTTTC

Primer name: sequence (50 –30 )

Fungal species

Table 1 (continued )

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

dCTP should be routinely advised in the PCR of extremely A+T-rich sequences. 2.2. Use of anonymous unique DNA regions for designing PCR primers 2.2.1. Sequence characterized amplified region Species-specific sequence characterized amplified region (SCAR) markers have been widely used for the development of PCR primers (Table 2). After comparing randomly amplified polymorphic DNA (RAPD) markers of a target pathogen with those of non-target organisms, unique bands specific to the target pathogen could be observed and cloned. Once unique bands have been detected, they are usually used as probes to check the presence of similar DNA sequences in related species. If the DNA fragment does not cross-react, it can be sequenced, and the sequence of this SCAR marker can be used for designing speciesspecific PCR primers. Specificities of primers are further tested using a number of isolates of the target taxon, preferably from a wide geographical range, and a number of closely related nontarget taxa. Because short primers (10–12 bases in length) are used, RAPD-PCR amplifications require low annealing temperature (35–37 1C), which are not stringent and increase the chance of nonspecific priming. Sometimes, RAPD markers are difficult to be reproduced between laboratories and even within laboratories. Recently, microsatellite primed-PCR (MP-PCR) and repetitive PCR (rep-PCR) are considered more robust than conventional RAPDs, because longer primers are used for MP-PCR and rep-PCR as compared to RAPDs. This allows more stringent annealing temperatures and reaction conditions that enhance reproducibility (Ma et al., 2001). Microsatellite and repetitive sequence regions with high numbers of copies are widely dispersed in eukaryotic genomes, including plant pathogenic fungi (Ma et al., 2001). Thus, these regions have a potential for developing highly sensitive PCR primers for many phytopathogenic fungi. Here is an example for developing M. fructicola-specific PCR primers based on a MP-PCR marker. In a study on population structure of M. fructicola, we observed that the microsatellite primer M13 (50 -GAGGGTGGCGGTTCT-30 ) amplified a DNA fragment of approximately 740-bp in size from each of more than 500 isolates of M. fructicola collected worldwide. Although the M13 primer also amplified an approximately 740-bp fragment from M. laxa, the nucleotide sequences of the fragments from M. fructicola and M. laxa showed significant difference with only 42% similarity. Based on the sequences of the fragments, we developed M. fructicola-specific nested PCR primer pairs, which were sensitive enough to detect two conidial spores of M. fructicola per PCR amplification (Ma et al., 2003b). The highly sensitive PCR assay with these primer pairs has been used to monitor the airborne inoculum of M. fructicola in stone fruit orchards in California.

151

2.2.2. Cloned repetitive sequences Characterized cloned repetitive sequences are also useful targets for the development of PCR primers (Table 2). Repetitive chromosomal DNA sequences comprise a significant fraction of many eukaryotic genomes and have been shown to be highly variable at specific level. Since they are presumed to be non-coding and thus selectively neutral, these sequences have formed the basis for the development of informative, species-specific diagnostic markers (Boehm et al., 2001). Normally, repetitive species-specific clones could perhaps be found simply by screening either a partial plasmid library or complete lambda library with total labeled genomic DNA as a probe. Since repetitive sequences are over represented as compared to single copy sequences, only the former can generate strong signals under stringency hybridization conditions. Subsequently, species-specific clones could be tested by the use of DNA from a range of related fungal species. Finally, PCR primers could be designed based on sequences of the species-specific clones. For example, in a study on PCR detection of M. fructicola, Boehm et al. (2001) identified three clones of species-specific repetitive sequences from a partial library having 312 recombinant clones. The species-specific clones hybridized to 60 geographically diverse M. fructicola isolates. Based on the sequences of the species-specific clones, we developed species-specific primer pairs, which were sensitive enough to detect 10 spores of M. fructicola (Boehm et al., 2001). Since the repetitive DNA sequences are repeated ranging from moderately to a very high number of copies, primers developed based on repetitive sequences should be much more sensitive than those developed from single copy genes in theory. 2.3. Design of degenerate PCR primers for amplification of homologous sequences Degenerate primers are particularly useful in amplifying homologous genes from different organisms because a known gene in one organism may have its homolog sequence in other genetically related organisms. Homologous genes display regions where they are highly conserved and also regions where they have evolved and are divergent. Degenerate primers can be designed based on the sequences of highly conserved regions. Currently, a free computer program Primaclade (http://www.umsl.edu/ services/kellogg/primaclade.html) is available for design of degenerate PCR primers. Primaclade is a web-based application that accepts a multiple species nucleotide alignment file as input and identifies a set of PCR primers that will bind across the alignment. Basically, there are four steps for designs of degenerate PCR primers with this program: (1) obtain candidate genes from the GenBank or other databases; (2) align the sequences of candidate genes; (3) input the alignment file into Primaclade program; and (4) run the program to get a number degenerated primers. From the list of primers, you can select a forward

Pa2071: GGGCGTTATGCGAGATCAGG Pa2072: GTATTTGTAGGAATTTCCAG

OPC7-FS-30: GTCCCGACGACAACACCAAGAAAGACAACG OPC7-RS-25: GTCCCGACGAGGTTGGTGGCAAGTG

Alternaria radicina

Aphanomyces euteiches

Nested-PCR External primer pair EBdF: CCCCGGCAGTCAGTGCAAGGC EBdR: GTTTCGGGTATCCCGCACACCATGG

Botryosphaeria dothidea

OPT18F470: GATGCCAGACCAAGACGAAG OPT18R470: GATGCCAGACGCACTAAGAT

Fc01F: ATGGTGAACTCGTCGTGGC Fc01R: CCCTTCTTACGCCAATCTCG

Fg16F: CTCCGGATATGTTGCGTCAA Fg16R: GGTAGGTATCCGACATGGCAA

Fcg17F: TCGATATACCGTGCGATTTCC Fcg17R: TACAGACACCGTCAGGGGG

53-6F: TTTACGAGGCGGCGATGGGT 53-6R: GGCCGTTTACCTGGCTTCTT

FUS1: CTTGGTCATGGGCCAGTCAAGAC FUS2: CACAGTCACATAGCATTGCTAGCC

Fusarium culmorum

F. culmorum

F. graminearum

F. culmorum and F. graminearum

F. moniliforme

F. moniliforme

Repetitive genomic clone

SCAR

SCAR

SCAR

SCAR

SCAR

350

1600

561

340

400–500

570

472

450

SCAR

SCD18A: GAGTACGTTGGTACAATGG SCD18B: ACTCTCTCTCGTCTTTTGC

SCA10A: TAGTGGTGTCAGTGAAAGG SCA10B: TGCTAAAGCTTAAAATCCC

Eutypa lata

413

UP-PCR markerd

700

BA2f: GTGGGGGTAGGATGAGATGATG BA1r: TGAGTGCTGGCGGAAACAAA

Botrytis spp. (B. aclada, B. allii; B. byssoidea; B. squamosa; and B. cinerea)

700

SCAR

SCB02A: AATCGATGTGAGAGATGG SCB02B: AGGTCAATGATAGCCAAC

C729+: AGCTCGAGAGAGATCTCTGA C729: CTGCAATGTTCTGCGTGGAA

Botrytis cinerea

701

MP-PCR markerc

627

260

AFLP markerb

718

1322

900

SCARa SCAR

Product size (bp)

Target DNA

55

61

Touchdown PCR

Touchdown PCR

Touchdown PCR

55

60

60

60

60

60

68

68

55

72

72

60

Annealing temperature (1C)

Murillo et al. (1998)

Moller et al. (1999)

Nicholson et al. (1998)

Nicholson et al. (1998)

Nicholson et al. (1998)

Schilling et al. (1996)

Lecomte et al. (2000)

Nielsen et al. (2002)

Rigotti et al. (2002)

Ma et al. (2003b)

Schmidt et al. (2003)

Vandemark et al. (2000)

Pryor, & Gilbertson (2001)

Reference

152

Internal primer pair IBdF: CTGCATGAACCAATGTCCGAC IBdR: AGGATGGAGAGCACAGTCCGT

OCA-V: ATACCACCGGGTCTAATGCA OCA-R: TGCCGACAGACCGAGTGGATT

Aspergillus ochraceus

OPB10-FS-25: CTGCTGGGACATCCAGGAATGAGAC OPB10-RS-25: CTGCTGGGACCAAATCGCGTGCAGG

Primer name: sequence (50 –30 )

Fungal species

Table 2 PCR primers developed based on sequences of anonymous unique DNA regions for the detection of phytopathogenic fungi

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

330

Bik3: GTTCCTACGGATAAGACC Bik4: TTTGACCAAGATAGATGCC

A280: TATACCGGACGGGCGTAGTGACGATGG B310: CAGCCATTCATGGATGACATAACGAATTTC

61-1F: GGCCACTCAA GAGGCGAAAG 61-1R: GTCAGACCAGAGCAATGGGC

D1: GCGTAAGAAGCGTGCCTTAGAGTC D2: TCCTGCTCCTACTCCTTCTCTAGC

Y13MF: CTTGAGGCGGAAGATCGC Y13MR: ATCCCTTTTCCGGGGTTG

Y13NF: ACCAGCCGA TTTGTGGTTATG Y13NR: GGTCACGAGGCAGAGTTCG

Nested-PCR External primer pair, EMfF: ACAACGAGAGCTTTCTGTAAGAATTCCATCA EMfR: ACGTATATGATCCCTCCAACATCGTTGA

F. oxysporum f. sp. phaseoli

F. subglutinans

Leptosphaeria maculans (A-type)

Microdochium nivale var. majus

M. nivale var. nivale

Monilinia fructicola

Andean group-specific primer pair, Pa3093: CAATCGCCGTACATGACTAA Pa3185: CCGTTACCTCTATATTCCCAA

Phaeoisariopsis griseola

Mesoamerican group-specific primer pair, Pm2981: CAATCGCCGTTTACGAAGAT Pm2982: CAATCGCCGTCGATCGATGA

X-09intF3: GATTCAGCTTGTGCGTAT X-09R: GGTCTGGTTGGTATACC

M. fructicola M. laxa

Internal primer pair, IMfF: ATGCAGAAGTGTGAATAGGGCCT IMfR: CGAAGGATGAGAGGAAGATTAGGG

Multiplex PCR Primer A: CATCTCACGACAACATTAGGG Primer B: AAGCTCACGAAAGTTGGTGGG and Primer E: CAGCTCACGACCTGTAGT Primer F: CAGCTCACGATGGGAATC

Internal primer pair, WiltNF-2: TTGTATGGCGTTGGAGAGGG WiltNR-2: TTGTTCAGATCGGAATCGGG

SCAR

SCAR

MP-PCR marker

SCAR

SCAR

Repetitive sequence

SCAR

SCAR

SCAR

SCAR

382

Bik1: ATTCAAGAGCTAAAGGTCC Bik2: AAAGGTAGTATATCGGAG

Nested PCR External primer pair, WiltNF-1: ATAGCCAAGCCGACCCTCAC. WiltNR-1: ACGAGGTTCGTCGTTGTTC

SCAR

Bik1: ATTCAAGAGCTAAAGGTCC Bik4: TTTGACCAAGATAGATGCC

F. oxysporum f. sp. gladioli race 1

F. oxysporum f. sp. ciceris

F. oxysporum f. sp. basilici

700

400

500

468

571

300

220

~600

445

609

1196

609

605

849

943

62

63

55

64

64

62

62

72

64

65

60

Touchdown PCR

65 or touchdown PCR

Touchdown PCR

Touchdown PCR

Touchdown PCR

Guzma´n et al. (1999)

Forster and Adaskaveg (2000)

Ma et al. (2003b)

Nicholson et al. (1996)

Nicholson et al. (1996)

Calderon et al. (2002)

Moller et al. (1999)

Alves-Santos et al. (2002)

de Haan et al. (2000)

Garcia-Pedrajas et al. (1999)

Chioccetti et al. (2001)

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161 153

PSB12-FS-24: CCTTGACGCACAGAGAATGATTGG PSB12-RS-26: CCTTGACGCATGTTAGCATAAAATCC

CAMB3: GTGACGTAGGTTCATCTGCT CAMB4: GTGACGTAGGCCAAAATAACA

Real-time PCR P3: GCCAT CAAGACGTGCGAGA P4: GGCAGGGATTCGGGCATA Probe F2: AGAGTAAGTATATTTTGCCCGACCCCGCCTp

O8-1: AAGATGATGTTGGATGATTG O8-2: TGCCTGATTTCTACCTTCT

Phoma sclerotioides

Phytophthora cambivora

P. infestans

P. infestans, P. mirabilis, and P. phaseoli

p990F:d-GGTGGGTGGAACGAAGGA p1050R: d-TGGCAGCGGAGATCCAA p1010CT:CCGCGCCAGTATTTGTCTTCCGG

Nested PCR External primer pair, QUERC1: GTGATCGCAGGAGTGCTCTT 3 QUERC2: GTGATCGCAGTAAGAAATGAGT

P. medicaginis

P. quercina

SIRO1: TGCCGAGCTGACGGGCCTC SIRO6: GCCGAGCTGTCTACAGAGAA

SR1F: CAGGTTATGTATGGGCCG SR1R: TTGAGCGATGACCATTCC

Sirococcus conigenus

Sporisorium reiliana

Ustilago maydis

PG2F: CTTCTTAGCTGGGGCTACCGTC PG2R: ACCGACTCGGGAAAAGAGCA

Pyrenophora graminea

UM11F: GAACCTTTCTGGCCTCCTTT UM11R: CCTTGGTTTCCGTTCCGTAC

SR3F: GCAGCCTCAGCATTACTC SR3R: ATACACCTGTGACGGCTG

8MREV: GTCACACCATGCACATCTCGTGTG 8MFW: CAATCCGATGGAAGTTGACGGCGT

Plasmopara halstedii

Internal primer pair, QUERC3: GAGTGCTCTTTAGTGTCGAC QUERC4: GAAATGAGTGTGATCCATTCCA

AE7-1: GCCGCCGACATATTGAAT AE7-2: CAAATCTGCGAACGAGACAT

P. infestans, P. mirabilis, and P. phaseoli

Repetitive sequence of genomic DNA

Repetitive sequence of genomic DNA

SCAR

SCAR

SCAR

SCAR

SCAR

Repeated DNA sequence

Repeated DNA sequence

Species-specific GC-rich nuclear satellite DNA region

SCAR

SCAR

Target DNA

900

680

960

944

435

1038

819

842

61

171

258

245

73

1,105

499

Product size (bp)

56

56

66

68

50

58

Touchdown PCR

60

50

50

50

60

62

70

Annealing temperature (1C)

Xu et al. (1999)

Xu et al. (1999)

Bahnweg et al. (2000)

Taylor et al. (2001)

Roeckel-Drevet et al. (1999)

Nechwatal et al. (2001)

Vandemark, & Barker (2003)

Judelson& Tooley (2000)

Judelson& Tooley (2000)

Bohm et al. (1999)

Schubert et al. (1999)

Larsen et al. (2002)

Reference

154

O8-3: GAAAGGCATAGAAGGTAGA O8-4: TAACCGACCAAGTAGTAAA

Primer name: sequence (50 –30 )

Fungal species

Table 2 (continued )

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

b

SCAR: sequence characterized amplified region. AFLP: amplified fragment length polymorphism. c MP-PCR: microsatellite primed-PCR. d UP-PCR: universally primed-PCR.

a

Vfa-F: CGGCAGGTATGTTTGCACAATC Vfa-R: CCGCACCTTACGATTAGAAGTC

Verticillium fungicola SCAR

SCAR 162

521

68

67

64

824

VDS1: CACATTCAGTTCAGGAGACGGA VDS2: CCTTCTACTGGAGTATTTCGG

64

1163

58

64

58

Internal primer pair, INTNDf: CCACCGCCAAGCGACAAGAC INTNDr: TAAAACTCCTTGGGGCCAGC or INTND2f : CTCTTCGTACATGGCCATAGATGTGC INTND2r: CAATGACAATGTCCTGGGTGTGCCA

1410

462

548

SCAR

SCAR

Nested-PCR External primer pair, NDf: ATCAGGGGATACTGGTACGAGA NDr: GAGTATTGCCGATAAGAACATG

Internal primer pair, INTD2f: ACTGGGTATGGATGGCTTTCAGGACT INTD2r: TCTCGACTATTGGAAAATCCAGCGAC

Nested-PCR External primer pair, D1: CATGTTGCTCTGTTGACTGG D2: GACACGGTATCTTTGCTGAA

V. dahliae

Verticillium dahliae nondefoliating pathotype

Verticillium dahliae defoliating pathotype

Romaine et al. (2002)

Li et al. (1999)

Mercado-Blanco et al. (2001)

Mercado-Blanco et al. (2002)

ARTICLE IN PRESS

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161 155

ARTICLE IN PRESS 156

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

degenerate primer at 50 end region and a reverse degenerate primer at 30 end region, both located at DNA sequence encoding conserved amino acids. Using the degenerate PCR primers, a homologous DNA fragment could be amplified from the target species. Based on the DNA sequence of the amplified fragment, a pair of speciesspecific primers can be designed for the target species. 2.4. Primers for multiplex PCR Most current molecular diagnostic assays used in plant pathology target one specific pathogen. However, because crops can be infected by numerous pathogens, it is desirable to develop assays that can detect multiple pathogens simultaneously (Lievens & Thomma, 2005). Multiplex PCR has the potential to produce considerable savings of time, since in a multiplex PCR amplification more than one target sequences are amplified. Because of more than one pair of PCR primers used in single reactions, however, design of compatible PCR primers without interference is critical for a successful multiplex PCR analysis. This may be achieved through the utilization of primers with nearly identical optimum annealing temperatures and should not display significant homology either internally or to one another. The presence of more than one primer pair in a multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of primer dimers. These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components (Elnifro et al., 2000). Thus, the optimization of multiplex PCR conditions should aim to minimize such nonspecific amplifications. Optimization of PCR components such as PCR buffer constituents, dNTPs, concentrations of enzyme, primers and DNA template in multiplex PCRs has been proven to be beneficial. Additionally, PCR additives, such as dimethyl sulfoxide, glycerol, bovine serum albumin, or betaine, have been reported to be of benefit in multiplex PCRs. But empirical testing and a trialand-error approach have to be used during the optimization of multiplex PCR conditions, because there are no means to predict the performance characteristics of a selected primer pair in each case. Prior to application of a multiplex PCR in testing real samples, the multiplex PCR must be evaluated for its sensitivity as compared with the corresponding uniplex PCRs because preferential amplification of one target sequence over another (bias in template-to-product ratios) is a common phenomenon in multiplex PCRs, which may decrease sensitivity of the muliplex PCR in detecting some of target fungi. 3. Real-time PCR for the detection of phytopathogeni fungi While the conventional PCR method has revolutionized the detection of plant pathogens, the quantitative relationship between the amount of starting template DNA and the amount of PCR product at any cycle has been difficult to

assay with traditional end point PCR methods. The recent development of real-time quantitative PCR has eliminated the limitation of conventional PCR, thus allowing the routine and reliable quantification of PCR products. Unlike conventional end-point quantitative PCR, real-time PCR monitors PCR products as they accumulate in the exponential phase, before reaction components become limiting. Since the measurement of fluorescence through the reaction by a fluorometer eliminates the need for postPCR processing steps, an accurate diagnosis can be made in less than 2–5 h with real-time PCR. Thus a significant improvement introduced by real-time PCR is the increased speed for pathogen detection. This is primarily due to the reduced cycle times, removal of separate post-PCR detection procedures, and the use of sensitive fluorescence detection equipment allowing earlier amplicon detection (Mackay, 2004). One drawback of real-time PCR is the relatively high cost of the equipment. There are currently several fluorescence chemistry formats, SYBRTM green I, TaqManTM, molecular beacons, and ScorpionTM, have been developed to monitor amplified products during amplification, each with its advantages and disadvantages. SYBR green I is the simplest and the less expensive system. But this single-dye chemistry is limited for detecting multiple targets in a multiplex real-time PCR. TaqMan system makes use of two primers plus an oligonucleotide that hybridizes to a sequence within the amplicon. TaqMan probes contain a fluorescent reporter dye with a quencher dye in close proximity to reduce the fluorescent signal. During PCR, the fluorogenic probe binds to its target sequence, within the DNA fragment being amplified. As the PCR process continues, the 50 nuclease activity of DNA polymerase cleaves the probe, thus separating the reporter and quencher dyes and causing an increase in fluorescence proportional to the amount of PCR product present. Compared to the SYBR green I system, the TaqMan system is more specific because both the probe and primers are specific to target sequences. Other types of fluorescent probes and primers, including molecular beacons and Scorpion have also been used in real-time PCR assays. Molecular beacons offer very low background fluorescence, extended dynamic range, and the ability to distinguish two targets on the basis of a single-nucleotide difference. Scorpion chemistry is reported to be more rapid and efficient than TaqMan and molecular beacons (Okubara et al., 2005). Primers designed for use in conventional PCR can be utilized in SYBR green I real-time PCR assays if amplicon size criteria are met. Primer design software such as Primer3, Primer Express (PE Applied Biosystems, Foster City, CA), and Real-time PCR Primer Design (https:// www.genscript.com/ssl-bin/app/primer) are widely used for designing TaqMan primers and probes. The technical aspects of primer and probe design have been reviewed elsewhere (Ginzinger, 2002). Although primers and probes designed by such programs are predicted to be specific to

ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

target sequences, the primers and probes must be tested using actual samples and a range of nontarget species. It is necessary to mention that although most real-time PCR assays in themselves are characterized by high precision and reproducibility, the accuracy of the obtained data is largely depended on several other factors such as sample preparation, quality and stability of the standard curve. Therefore, the accuracy of the obtained data needs to be checked during the development of the assay by comparison with other established assays. This is the major task for molecular diagnostics to guarantee reliable data (Klein, 2002). 4. Approaches for eliminating PCR inhibitors For PCR detection of phytopathogenic microorganisms, the choice of DNA extraction methods depends on the type of sample. For identification of a pathogen from cultures, in most cases, it is possible to obtain qualifying template DNA for PCR amplifications by simply using a mycelial boiling method. Briefly, fungal mycelia or spores (10–100 mg) are collected from a culture or from diseased plant tissues, and placed into a 1.5-ml microcentrifuge tube containing 20–100 ml of 1 M Tri-HCl (pH 8.0) overlaid with 2 drops of mineral oil. Then, samples are boiled at 98–100 1C for 15 min and immediately placed on ice for 5 min. After the tubes are centrifuged for 2 min, a 1–2 ml aliquot of resulting liquid of each sample could be used for PCR amplification (Rollo et al., 1990; Ma and Michailides, 2002; Ma et al., 2003c). To effectively detect pathogens in infected plant tissues, in soil, air, or water samples, however, suitable DNA extraction procedures are necessary in order to eliminate PCR inhibitors released from these samples. 4.1. Plant tissue samples Boiling procedures described above may also be used for PCR detection of some pathogens in infected plant materials, such as Gremmeniella abietina in infected twigs of pine (Hamelin et al., 2000) and Eutypa lata in infected grapevine wood tissues (Lecomte et al., 2000). More often, however, DNA must be extracted from infected plant tissue in order to effectively detect small amount of fungal DNA in these samples. Furthermore, even when DNA was extracted successfully from these samples, it might still pose difficulty in using it as template for PCR amplifications because of PCR inhibitors. Thus, a set of primers that amplify plant DNA as a control is needed to prove that the extracted DNA is amplifiable (Tooley et al., 2006). In plant tissues, various compounds (including polysaccharides and phenolic compounds) can inhibit PCR amplifications (Wilson, 1997). Some of these inhibitors could inhibit PCR amplification by chelating the Mg2+ cofactor for Taq polymerase, or by binding to target DNA or DNA polymerase, and thus interfering in the reaction between the polymerase and target DNA (Wilson, 1997).

157

To attenuate the effects of PCR inhibitors, dilution of DNA extracts has been proven to eliminate effects of PCR inhibitors in many studies, but it causes a reduction in PCR sensitivity. Commercial DNA extraction kits can remove most PCR inhibitors efficaciously, but not all inhibitors in some specific cases. Thus, additions of amplification facilitators, such as citric acid, sodium sulfite, polyvinylpyrrolidone (PVP), polyvinyl polypyrrolidone (PVPP), BLOTTO (10% skim milk powder and 0.2% NaN3), dimethyl sulfoxide (DMSO), skim milk, bovine serum albumin (BSA), or T4 gene 32 protein (gp32) to DNA extraction buffers or to PCR mixture have been documented in literature (Wilson, 1997; Singh et al., 1998; GarciaPedrajas et al., 1999; Louws et al., 1999; Singh et al., 2002). The addition of 0.65–0.70% of sodium sulfite in the DNA extraction buffer could minimize the inhibition effects of polyphenolics and improve the PCR detection of plant pathogens in potato tubers and in sweet cherry leaves and barks (Singh et al., 2002). The incorporation of 1.2% of citric acid in the DNA extraction buffer was found to prevent chlorogenic acid in potato tissue from inhibiting PCR amplifications (Singh et al., 1998). Poussier et al. (2002) reported that the addition of 2% PVP or PVPP to the DNA extraction buffer increased significantly the PCR detection of phytopathogenic pathogens in tomato, eggplant, and pepper, but the combination of the addition of 5% PVPP to DNA extraction buffer and 500 ng or 5 mg of (BSA) to the PCR mixture gave the best amplification. Additions of PVPP, sodium ascorbate, and hexadecyltrimethylammonium bromide (CTAB) in cell lysis buffer can partially remove humic compounds derived from soil samples (Robe et al., 2003). In addition to inclusions of the above reagents to DNA extraction buffers, various reagents have been also added to PCR mixture buffers by different researchers to circumvent the inhibition problem. In order to detect Alternaria radicina on carrot seed efficiently, Pryor and Gilbertson (2001) found that when 0.2% skim milk was included in the PCR mixture, the target fungal DNA was consistently amplified from undiluted DNA extraction obtained from infected seeds. The addition of 4% BLOTTO, 0.8% (wt/vol) PVP and 0.8% (wt/vol) ficoll, or only PVP at 1–2% in PCR reaction buffers could avoid the inhibitory effects of polyphenolic and other compounds derived from plant tissues. Additionally, the use of DMSO and glycerol at concentrations varying between 5–10% (vol/vol) could also improve PCR efficiency and specificity. Henegariu et al. (1997) found that BSA, at concentrations of up to 0.8 mg/ml, increased efficiency of PCR amplification much better than DMSO or glycerol. Although various reagents have been used to eliminate the effects of PCR inhibitors by different researchers in different detection systems, it should be noted that the usefulness and the optimal concentrations of such ‘adjuvants’ have to be tested in each case since some of these reagents might also inhibit PCR amplifications when they are used at high concentrations (Wilson, 1997; Koonjul et al., 1999).

ARTICLE IN PRESS 158

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

4.2. Air samples Spore traps are conventionally used to determine the spore density for air-borne disease agents. Microscopic examination of spores collected on spore-trap tapes is a time-consuming method and requires special training. Previous studies have shown that fungal DNA could be extracted from samples taken by a Burkard spore-traps (used to trap airborne plant pathogens) and detected by PCR. To remove spores from the tapes for DNA extraction, each spore-trap tape section can be cut into few small pieces and put into a 2-ml microcentrifuge tube containing 1.5 ml of 0.1% Nonidet. The tubes are incubated at 55 1C for 10 min, shaken for 10 min. Then the tubes are spun at 10,000g for 10 min, and the supernatants are decanted. Disruption of spores can be achieved by shaking spore suspensions for two to five times of 40 s in a FastPrep FP120 Cell Disrupter (Qbiogene, Carlsbad, CA). Then, extraction of DNA from the spores can be performed using commercial DNA extraction kits (such as the FastDNA Kit), according to the manufactures’ instructions. For dirty air samples, a second purification using a commercial kit may be required to remove residual PCR inhibitors in the first round of DNA extract. The final DNA is dissolved in 5–50 ml of H2O and 5-ml aliquots of 1:10 DNA dilutions are used for PCR amplifications. Using this method, we successfully detected as few as 30 spores of M. fructicola on a 48-mm piece of spore-trap tape. 4.3. Soil samples Two approaches have been developed for extracting DNA from soil samples. One is the direct extraction of DNA from soil samples after in situ cell lysis followed by DNA purification. Currently, physical, chemical, and enzymatic cell disruption are three major types of cell lysis used alone or in combinations. Physical disruption is achived by freezing–thawing or freezing–boiling, and beadmill homogenization treatments. These physical methods are effective to disrupt fungal mycelia and spores but they often result in significant DNA shearing. Chemical lysis either alone or in association with physical methods has been used extensively. Probably the most common chemical is the detergent sodium dodecyl sulfate (SDS), which dissolves the hydrophobic material of cell membranes. Additionally, amplification facilitators (such as PVP, PVPP, and skim milk) may also be used in cell lysis buffers to reduce effects of humic acids from soil samples on inhibiting PCR. Lysozyme and proteinase K are the most common enzymes used in enzymatic cell disruption methods. The second approach separates the microbial cells from soil particles by differential centrifugation. Then the separated cells are lyzed and used for nucleic acid purification. After cell lysis, a nucleic acid purification step always has to be conducted since the humic acid in soil inhibits polymerase chain reactions. In most studies,

following cell lysis, nucleic acids are purified by organic solvent extraction (either phenol or chloroform), and precipitated by ethanol or isopropanol. Currently, although various commercial kits (FastDNA SPIN kit for soil, UltraClean soil kit, QIAamp DNA stool minikit, and QIAamp DNA minikit) have been developed for DNA extraction from soil samples, in some cases, a second round of DNA purification may still be necessary to remove residual humic acid. 4.4. Water samples Extraction of high-quality DNA is a key step in PCR detection of pathogen in water samples. In general, pathogens in water samples have to be concentrated before DNA extraction. Centrifugation and filtration are the current two primary approaches for concentration of pathogen. For the centrifugation approach, most fungal spores can be collected by centrifuging water samples at 10,000g for 10 min and using the resultant pellet for DNA extraction with a commercial kit. But in most studies, a filtration method was widely used since this method is appropriate for the elimination of PCR inhibitors such as organic and inorganic compounds present in water samples. Hong et al. (2002) evaluated nine hydrophilic membranes for recovery of pythiaceous species from water samples and found that Durapore5 membrane (Millipore Corporation, Bedford, MA) not only increased the sensitivity of filter-based isolation for quantifying pythiaceous species in water but also saved filtering time. Following the filter-based spore collections, the filter can be cut into fine pieces and directly used for DNA extraction. Additionally, to further eliminate PCR inhibitors, various amplification facilitators described above or anti-inhibitory substance treatments, such as GeneReleaser or ultrafiltration treatment before PCR may also be helpful for PCR detection of pathogen in water samples. Jiang et al. (2005) reported that the effect of PCR inhibitors released from water samples could be relieved significantly by the addition of 400 ng of bovine serum albumin/ml or 25 ng of T4 gene 32 protein/ml to the PCR mixture (Jiang et al., 2005). 5. Conclusions Molecular diagnostics have impacted considerably on research in plant pathology, although they are still in their infancy. The use of PCR methods to detect pathogens in plant tissues or other samples will be very effective once the problems of PCR inhibitors are overcome. Several approaches have been used to design species-specific PCR primers and the sequence of rDNA is the commonest primer target in PCR diagnosis of phytopathogenic fungi. Additionally, anonymous unique DNA regions generated by RAPD, MP-PCR, or rep-PCR have also gained widespread usage in development of PCR primers specific to target pathogens. Commonly used methods to overcome

ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

PCR inhibitors include the use of commercial DNA extraction kits combined with additions of amplification facilitators in DNA extraction and PCR reaction buffers. Acknowledgments Part of the research reviewed in this manuscript was supported USDA Award no. 2002-51100-01990, the University of California Specialty Crop Research Program (Project no. SA6677), and National Science Foundation of China (30500334). Reference Alves-Santos, F.M., Ramos, B., Garcia-Sanchez, M.A., Eslava, A.P., Diaz-Minguez, J.M., 2002. A DNA-based procedure for in planta detection of Fusarium oxysporum f. sp. phaseoli. Phytopathology 92, 237–244. Bahnweg, G., Schuber, R., Kehr, R.D., Muller-Starck, G., Heller, W., Langebartels, C., Sandermann, H.J., 2000. Controlled inoculation of Norway spruce (Picea abies) with Sirococcus conigenus, PCR-based quantification of the pathogen in host tissue and infection-related increase of phenolic metabolites. Trees 14, 435–441. Boehm, E.W.A., Ma, Z., Michailides, T.J., 2001. Species-specific detection of Monilinia fructicola from California stone fruits and flowers. Phytopathology 91, 428–439. Bohm, J., Hahn, A., Schubert, R., Bahnweg, G., Adler, N., Nechwatal, J., Oehlmann, R., Osswald, W., 1999. Real-time quantitative PCR, DNA determination in isolated spore of the mycorrhizal fungus Glomus mosseae and monitoring Phytophthora infestans and Phytophthora citricola in their respective host plants. J. Phytopathol. 147, 409–416. Calderon, C., Ward, E., Freeman, J., Foster, S.J., McCartney, H.A., 2002. Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays. Plant Pathol. 51, 303–310. Chioccetti, A., Bernardo, I., Daboussi, M.J., Garibaldi, A., Gullino, M.L., Langin, T., Migheli, Q., 1999. Detection of Fusarium oxysporum f. sp. dianthi in carnation tissue by PCR amplification of transposon insertions. Phytopathology 89, 1169–1175. Chioccetti, A., Sciaudone, L., Durando, F., Garibaldi, A., Migheli, Q., 2001. PCR detection of Fusarium oxysporum f. sp. basilici on basil. Plant Dis. 85, 607–611. de Haan, L.A.M., Numansen, A., Roebroeck, E.J.A., van Doorn, J., 2000. PCR detection of Fusarium oxysporum f. sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms. Plant Pathol. 49, 89–100. Dieffenbach, C.W., Lowe, T.M.J., Dveksler, G.S., 1993. General concepts for PCR primer design. In: PCR Methods and Applications, Cold Spring Harbor Laboratory, vol.3, pp. S30–S37. Dyer, P.S., Furneaux, P.A., Douhan, G., Murray, T.D., 2001. A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis. Fungal Genet. Biol. 33, 173–180. Elnifro, E.M., Ashshi, A.M., Cooper, R.J., Klapper, P.E., 2000. Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 13, 559–570. Fernandez, D., Quinten, M., Tantaoui, A., Geiger, J.P., Daboussi, M.J., Langin, T., 1998. Fot1 insertions in the Fusarium oxysporum f. sp. albedinis genome provide diagnostic PCR targets for detection of the date palm pathogen. Appl. Environ. Microbiol. 64, 633–636. Ferreira, M.A.S.V., Tooley, P.W., Hatziloukas, E., Castro, C., Schaad, N.W., 1996. Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the karnal bunt of wheat fungus. Appl. Environ. Microbiol. 62, 87–93.

159

Filion, M., St-Arnaud, M., Jabaji-Hare, S.H., 2003. Direct quantification of fungal DNA from soil substrate using real-time PCR. J. Microbiol. Methods 53, 67–76. Forster, H., Adaskaveg, J.E., 2000. Early brown rot infections in sweet cherry fruit are detected by Monilinia-specific DNA primers. Phytopathology 90, 171–178. Foster, J., Singh, G., Fitt, B.D., Ashby, A.M., 1999. Development of PCR based diagnostic techniques for the two mating types of Pyrenopeziza brassicae (light leaf spot) on winter oilseed rape (Brassica napus ssp oleifera). Physiol. Mol. Plant Pathol. 55, 111–119. Fraaije, B.A., Lovell, D.J., Rohel, E.A., Hollomon, D.W., 1999. Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay. J. Appl. Microbiol. 86, 701–708. Fraaije, B.A., Lovell, D.J., Coelho, J.M., Baldwin, S., Hollomon, D.W., 2001. PCR-based assays to assess wheat varietal resistance to blotch (Septoria tritici and Stagonospora nodorum) and rust (Puccinia striiformis and Puccinia recondite) diseases. Eur. J. Plant Pathol. 107, 905–917. Frederick, R.D., Snyder, K.E., Tooley, P.W., Berthier-Schaad, Y., Peterson, G.L., Bonde, M.R., Schaad, N.W., Knorr, D.A., 2000. Identification and differentiation of Tilletia indica and T walkeri using the polymerase chain reaction. Phytopathology 90, 951–960. Garcia-Pedrajas, M.D., Bainbridge, B.W., Heale, J.B., Perez-Artes, E., Jimenez-Diaz, R.M., 1999. A simple PCR-based method for detection of the chickpea-wilt pathogen Fusarium oxysporum f. sp. ciceris in artificial and natural soil. Eur. J. Plant Pathol. 105, 251–259. Ginzinger, D.G., 2002. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512. Guzma´n, P., Gepts, P., Temple, S., Mkandawire, A.B.C., Gilbertson, R.L., 1999. Detection and differentiation of Phaeoisariopsis griseola isolates with the polymerase chain reaction and group-specific primers. Plant Dis. 83, 37–42. Hamelin, R.C., Bourassa, M., Rail, J., Dusabenyagasani, M., Jacobi, V., Laflamme, G., 2000. PCR detection of Gremmeniella abietina, the causal agent of Scleroderris canker of pine. Mycol. Res. 104, 527–532. Harmon, P.F., Dunkle, L.D., Latin, R., 2003. A rapid PCR-base method for the detection of Magnaporthe oryzae from infected perennial ryegrass. Plant Dis. 87, 1072–1076. Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H., Vogt, P.H., 1997. Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504–511. Hong, C., Richardson, P.A., Kong, P., 2002. Comparison of membrane filters as a tool for isolating pythiaceous species from irrigation water. Phytopathology 92, 610–616. Ioos, R., Frey, P., 2000. Genomic variation within Monilinia laxa, M. fructigena, and M. fructicola, and application to species identification by PCR. Eur. J. Plant. Pathol. 106, 373–378. Ito, S., Maehara, T., Maruno, E., Tanaka, S., Kameya-Iwaki, M., Kishi, F., 1999. Development of a PCR-based assay for the detection of Plasmodiophora brassicae in soil. J. Phytopathol. 147, 83–88. Jiang, J., Alderision, K.A., Singh, A., Xiao, L., 2005. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Appl. Environ. Microbiol 71, 1135–1141. Judelson, H.S., Tooley, P.W., 2000. Enhanced polymerase chain reaction methods for detecting and quantifying Phytophthora infestans in plants. Phytopathology 90, 1112–1119. Klein, D., 2002. Quantification using real-time PCR technology: applications and limitions. Tends Mol. Med. 8, 257–260. Kong, P., Hong, C.X., Jeffers, S.N., Richardson, P.A., 2003. A speciesspecific polymerase chain reaction assay for rapid detection of Phytophthora nicotianae in irrigation water. Phytopathology 93, 822–831. Koonjul, P.K., Brandt, W.F., Farrant, J.M., Lindsey, G.G., 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reverses the inhibitory effects of polyphenolic contamination of RNA. Nucleic Acid. Res. 27, 915–916.

ARTICLE IN PRESS 160

Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161

Lacourt, I., Duncan, J.M., 1997. Specific detection of Phytophthora nicotianae using the polymerase chain reaction and primers based on the DNA sequence of it elicitin gene ParA1. Eur. J. Plant Pathol. 103, 73–83. Larsen, R.C., Hollingsworth, C.R., Vandemark, G.J., Gritsenko, M.A., Gray, F.A., 2002. A rapid method using PCR-based SCAR markers for the detection and identification of Phoma sclerotioides: the cause of brown root rot disease of alfalfa. Plant Dis. 86, 928–932. Lecomte, P., Pe´ros, J.P., Blancard, D., Bastien, N., De´lye, C., 2000. PCR assays that identify the grapevine dieback fungus Eutypa lata. Appl. Environ. Microbiol. 66, 4475–4480. Li, K.N., Rouse, D.I., Eyestone, E.J., German, T.L., 1999. The generation of specific DNA primers using random amplified polymorphic DNA and its application to Verticillium dahliae. Mycol. Res. 103, 1361–1368. Li, S., Hartman, G.L., 2003. Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil. Plant Pathol. 52, 74–83. Lievens, B., Thomma, B.P.H.J., 2005. Recent developments in pathogen detection arrays: implications for fungal plant pathogens and use in practice. Phytopathology 95, 1374–1380. Louws, F.J., Rademaker, J.L.W., de Bruijn, F.J., 1999. The three Ds of PCR-based genomic analysis of phytobacteria: diversity, detection, and disease diagnosis. Annu. Rev. Phytopathol. 37, 81–125. Ma, Z., Michailides, T.J., 2002. A PCR-based technique for identification of Fusicoccum sp. from pistachio and various other hosts in California. Plant Dis. 86, 515–520. Ma, Z., Boehm, E.W.A., Luo, Y., Michailides, T.J., 2001. Population structure of Botryosphaeria dothidea from pistachio and other hosts in California. Phytopathology 91, 665–672. Ma, Z., Felts, D., Michailides, T.J., 2003a. Resistance to azoxystrobin in Alternaria isolates from pistachio in California. Pestic. Biochem. Physiol. 77, 66–74. Ma, Z., Luo, Y., Michailides, T.J., 2003b. Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachio in California. J. Phytopathol. 151, 312–322. Ma, Z., Yoshimura, M., Michailides, T.J., 2003c. Identification and characterization of benzimidazole resistance in Monilinia fructicola from stone fruit orchards in California. Appl. Environ. Microbiol. 69, 7145–7152. Ma, Z., Yoshimura, M., Holtz, B., Michailides, T.J., 2005. Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California. Pest Manage. Sci. 61, 449–457. Mackay, I.M., 2004. Real-time PCR in the microbiology laboratory. Clin. Microbiol. Infect. 10, 190–212. Martin, F.N., 1991. Selection of DNA probes useful for isolate identification of two Pythium spp. Phytopathology 81, 742–746. Martin, R.R., James, D., Levesque, C.A., 2000. Impacts of molecular diagnostic techniques on plant disease management. Ann. Rev. Phytopathol. 38, 207–239. Martin, F.N., Tooley, P.W., Blomquist, C., 2004. Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94, 621–631. Mayer, Z., Bagnara, A., Farber, P., Geisen, R., 2003. Quanti- fication of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. Int. J. Food Microbiol. 82, 143–151. McCartney, H.A., Foster, S.J., Fraaije, B., Ward, E., 2003. Molecular diagnostics for fungal plant pathogens. Pest Manage. Sci 59, 129–142. McKay, G.J., Egan, D., Morris, E., Brown, A.E., 1998. Identification of benzimidazole resistance in Cladobotryum dendroides using a PCRbased method. Mycol. Res. 102, 671–676. Mercado-Blanco, J., Rodriguez-Jurado, D., Perez-Artes, E., JimenezDiaz, R.M., 2001. Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR. Plant Pathol. 50, 609–619. Mercado-Blanco, J., Rodriguez-Jurado, D., Perez-Artes, E., JimenezDiaz, R.M., 2002. Detection of the defoliating pathotype of

Verticillium dahliae in infected olive plants by nested PCR. Eur. J. Plant Pathol. 108, 1–13. Mills, P.R., 1994. DNA-based methods for identification and characterization. In: Hawksworth, D.K.L. (Ed.), The identification and characterization of pest organisms CAB International, Egham. Surrey, UK, pp. 427–435. Moller, E.M., Chelkowski, 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. J. Phytopathol. 147, 497–508. Murillo, I., Cavallarin, L., Segundo, B.S., 1998. The development of a rapid for detection of Fusarium moniliforme. Eur. J. Plant Pathol. 104, 301–311. Nechwatal, J., Schlenzig, A., Jung, T., Cooke, D.E.L., Duncan, J.M., Osswald, W.F., 2001. A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P citricola in soil samples from oak stands. Forest Pathol. 31, 85–97. Nicholson, P., Lees, A.K., Maurin, N., Parry, D.W., Rezanoor, H.N., 1996. Development of a PCR assay to identify and quantify Microdochium nivale var nivale and Microdochium nivale var majus in wheat. Physiol. Mol. Plant Pathol. 48, 257–271. Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W., Joyce, D., 1998. Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol. Mol. Plant Pathol. 53, 17–37. Nielsen, K., Yohalem, D.S., Jensen, D.F., 2002. PCR detection and RFLP differentiation of Botrytis species associated with neck rot of onion. Plant Dis. 86, 682–686. Niessen, L., Vogel, R.F., 1997. Specific identification of Fusarium graminearum by PCR with GaoA targeted primers. Syst. Appl. Microbiol. 20, 111–123. Niessen, L., Vogel, R.F., 1998. Group specific PCR-detection of potential trichothecene producing Fusarium-species in pure cultures and cereal samples. Syst. Appl. Microbiol. 21, 618–631. Okubara, P.A., Schroeder, K.L., Paulitz, T.C., 2005. Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Can. J. Plant Pathol. 27, 300–313. Poussier, S., Cheron, J.J., Couteau, A., Luisetti, J., 2002. Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates. J. Microbiol. Methods 51, 349–359. Pryor, B., Gilbertson, R.L., 2001. A PCR-based assay for detection of Alternaria radicina on carrot seed. Plant Dis. 85, 18–23. Reischer, G.H., Lemmens, M., Farnleitner, A., Adler, A., Mach, R.L., 2004. Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe. J. Microbiol. Methods 59, 141–146. Rigotti, S., Gindro, K., Richter, H., Viret, O., 2002. Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers:Fr in strawberry (Fragaria  ananassa Duch) using PCR. FEMS Microbiol. Lett. 209, 169–174. Robe, P., Nalin, R., Capellano, C., Vogel, M.T., Simonet, P., 2003. Extraction of DNA from soil. Eur. J. Soil Biol., 183–190. Roeckel-Drevet, P., Tourvieille, J., Drevet, J.R., Says-Lesage, V., Nicolas, P., de Labrouhe, D.T., 1999. Development of a polymerase chain reaction diagnostic test for the detection of the biotrophic pathogen Plasmopara halstedii in sunflower. Can. J. Microbiol. 45, 797–803. Rollo, F., Salvi, R., Torchia, P., 1990. Highly sensitive and fast detection of Phoma traceiphila by polymerase chain reaction. Appl. Microbiol. Biotechnol. 32, 572–576. Romaine, C.P., Schlagnhaufer, B., Stone, M., 2002. A polymerase chain reaction-based test for Verticillium fungicola causing dry bubble disease on the cultivated mushroom, Agaricus bisporus. Appl. Microbiol. Biotechnol. 59, 695–699. Rondan-Duenas, J.C., Panzetta-Dutari, G.M., Gardenal, C.N., 1999. Specific requirements for PCR amplification of long mitochondrial A+T-rich DNA. Biotechniques 27, 258–260. Saiki, R.K., Scharf, S., Faloom, F., Mullis, K.B., Horn, G.T., Erlich, H.A., Arnheim, N., 1985. Enzymatic amplification of b-globin

ARTICLE IN PRESS Z. Ma, T.J. Michailides / Crop Protection 26 (2007) 145–161 genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1230–1350. Schaad, N.W., Frederick, R.D., 2002. Real-time PCR and its application for rapid plant disease diagnostics. Can. J. Plant Pathol. 24, 250–258. Schaad, N.W., Frederick, R.D., Shaw, J., Schneider, W.L., Hickson, R., Petrillo, M.D., Luster, D.G., 2003. Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues. Ann. Rev. Phytopathol. 41, 305–324. Schilling, A.G., Moller, 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, 515–522. Schmidt, H., Ehrmann, M., Vogel, R.E., Taniwaki, M.H., Niessen, L., 2003. Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP. Syst. Appl. Microbiol. 26, 138–146. Schubert, R., Bahnweg, G., Nechwatal, J., Jung, T., Cooke, D.E.L., Duncan, J.M., Muller-Starck, G., Langebartels, C., Sandermann, H., Osswald, W., 1999. Detection and quantification of Phytophthora species which are associated with root-rot disease in European deciduous forests by species-specific polymerase chain reaction. Eur. J. Forest Pathol. 29, 169–188. Schweigkofler, W., O’Donnell, K., Garbelotto, M., 2004. Detection and quantification of airbone conidia of Fusarium circinatum, the causal agent of pine pitch caker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method. Appl. Environ. Microbiol. 70, 3512–3520. Singh, R.P., Singh, M., King, R.R., 1998. Use of citric acid for neutralizing polymerase chain reaction inhibition by chlorogenic acid in potato extracts. J. Virol. Methods 74, 231–235. Singh, R.P., Nie, X., Singh, M., Coffin, R., Duplessis, P., 2002. Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR. J. Virol. Methods 99, 123–131. Smith, O.P., Peterson, G.L., Beck, R.J., Schaad, N.W., Bonde, M.R., 1996. Development of a PCR-based method for identification of Tilletia indica, causal agent of karnal bunt of wheat. Phytopathology 86, 115–122. Steenkamp, E.T., Wingfield, B.D., Coutinho, T.A., Zeller, K.A., Wingfield, M.J., Marasas, W.F.O., Leslie, J.F., 2000. PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex. Appl. Environ. Microbiol. 66, 4378–4382.

161

Su, X., Wu, Y., Sifri, C.D., Wellems, T.E., 1996. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 24, 1574–1575. Taylor, E.J.A., Stevens, J.A., Morreale, G., Lee, D., Kenyon, D.M., Thomas, J.E., 2001. Rapid-cycle PCR detection of Pyrenophora graminea from barley seed. Plant Pathol. 50, 347–355. Tooley, P.W., Goley, E.D., Carras, M.M., Frederick, R.D., Weber, E.L., Kuldau, G.A., 2001. Characterization of Claviceps species pathogenic on sorghum by sequence analysis of the beta-tubulin gene intron 3 region and EF-1 alpha gene intron 4. Mycologia 93, 541–551. Tooley, P.W., Martin, F.N., Carras, M.M., Frederick, R.D., 2006. Realtime fluorescent polymerase chain reaction detection of Phytophthora ramorum and Phytophthora pseudosyringae using mitochondrial gene regions. Phytopathology 96, 336–345. Vandemark, G.J., Barker, B.M., 2003. Quantifying Phytophthora medicaginis in susceptible and resistant Alfalfa with a real-time fluorescent PCR assay. J. Phytopathol. 151, 577–583. Vandemark, G.J., Kraft, J.M., Larsen, R.C., Gritsenko, M.A., Boge, W.L., 2000. A PCR-based assay by sequence-characterized DNA markers for the identification and detection of Aphanomyces euteiches. Phytopathology 90, 1137–1144. Wallenhammar, A.C., Arwidsson, O., 2001. Detection of Plasmodiophora brassicae by PCR in naturally infested soils. Eur. J. Plant Pathol. 107, 313–321. Wang, P.H., Boo, L.M., Lin, Y.S., Yeh, Y., 2002. Specific detection of Pythium aphanidermatum from hydroponic nutrient solution by booster PCR with DNA primers developed from mitochondrial DNA. Phytoparastica 30, 473–485. Ward, T.J., Bielawski, J.P., Kistler, H.C., Sullivan, E., Donnell, K., 2002. Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proc. Natl. Acad. Sci. USA 99, 9278–9283. White, T.J., Bruns, T., Lees, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Gelfland, D.H., Innis, M.A., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp. 315–322. Wilson, I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741–3751. Xu, M.L., Melchinger, A.E., Lubberstedt, T., 1999. Species-specific detection of the maize pathogens Sporisorium reiliana and Ustilago maydis by dot blot hybridization and PCR-based assays. Plant Dis. 83, 390–395.