Identification of Pathogens Directly from Plant Parts

Examples of studies using molecular methods to identify pathogens directly from plant parts are given in Table 2. Most of these involve identification of fungal species using RAPDs or PCR of the ITS-rDNA.

Developing methods for direct isolation of specific fungal DNA from plant tissues is more difficult than isolating DNA from a pure fungal strain, but the potential impact of the former methods is tremendous. These assays have demonstrated the presence of the pathogens in asymptomatic plants (Doohan et al. 1998). Some of these procedures take only seven to 24h to perform (Lee et al. 2001; Lovic et al. 1995), compared with several days to a week for traditional methods or methods requiring that the fungus be isolated prior to DNA

Table 1 Examples of studies on molecular identification of fungal plant pathogens in vitro

Fungal genus

Host

Level of discrimination

Method

Citation

Alternaria

Umbellifers

Species

RAPD

Pryor and Gilbertson 2002

Botrytis

Onion

Species subgroup

PCR-ITS/rDNA

Nielsen et al. 2001

Claviceps

Sorghum

Species/populations

AFLP and RAM

Tooley et al. 2000

Colletotrichum

Alfalfa

Species

AFLP

O'Neill et al. 1997

Elsinoe

Citrus

Species

RAPD

Hyun et al. 2001

Fusarium

Tomato

Virulence within race

RAPD

Mes et al. 1999

Fusarium

Cucumber

Forma specialis

RAPD

Vakalounakis and Fragkiadakis 1999

Gaeumannomyces

Turf-grass

Variety

PCR-ITS/rDNA

Goodwin et al. 1995

Gibberella (Fusarium)

Banana/corn

Species/toxicity/host

RAPD

Jimenez et al. 2000

Gibberella (Fusarium)

Banana/corn

Species

PCR-ITS/rDNA

Jimenez et al. 2000

Macrophomina

Bean, corn+

Population

AFLP

Mayek-Perez et al. 2001

Rhizoctonia

Various

Anastomosis grp subsets

PCR-ITS/rDNA

Carling et al. 2002

Rhynchosporium

Barley

Species

PCR-ITS/rDNA

Lee et al. 2001

Tilletia

Wheat

Species

TaqManPCR-MtDNA

Frederick et al. 2000

Venturia

Pear

Species

PCR-ITS/rDNA

Le Cam et al. 2002

Table 2 Examples of studies on molecular identification of fungal plant pathogens in vivo

Fungal genus

Host

Method

Level of discrimination

Citation

Alternaria

Carrot

PCR-ITS/rDNA

Species

Konstantinova et al. 2002

Fusarium

Wheat

RAPD

Species

Parry and Nicholson 1996

Fusarium

Wheat

RAPD

Species/variety

Doohan et al. 1998

Leptosphaeria

Crucifers

PCR/GenBank M77515a

Virulence

Taylor 1993

Melampsora

Willow

RAPD

Stem/leaf variants

Pei et al. 1997

Monosporascus

Cucurbits

PCR-ITS/rDNA

Species

Lovic et al. 1995

Monilinia

Stone fruits

dot blots and PCR

Species

Boehm et al. 2001

Mycosphaerella

Banana/plantain

PCR-ITS/rDNA

Species

Johanson and Jeger (1993)

Peronosclerospora

Sorghum

dot blots-genomic DNA

Species

Yao et al. 1990

Phakopsora

Soybean

TaqMan-PCR-ITS/rDNA

Species

Frederick et al. 2002

Pythium

1 ■ b 3 species

PCR-ITS/rDNA

Species

Kageyama et al. 1997

Rhynchosporium

Barley

PCR-ITS/rDNA

Species

Lee et al. 2001

aSeed cultured in liquid medium. b Cucumber, sugar beet, and Chinese cabbage.

aSeed cultured in liquid medium. b Cucumber, sugar beet, and Chinese cabbage.

extraction. Moreover, fungi that do not grow in pure culture may be studied with these techniques. On the other hand, amplifying DNA of nonviable fungi would lead to false-positives for disease potential, but would nevertheless provide useful information for researchers interested in mycotoxins.

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