Detection of grapevine trunk disease pathogens from field samples

Identification of the causal agent of a grapevine trunk disease based on the symptoms is often difficult due to the overlapping symptoms caused by different species and certain symptoms that closely

resemble those of other diseases such as Phomopsis dead arm and Eutypa dieback (Castillo-Pando et

al., 2001). The wedge-shape internal necrosis in the infected grapevine trunk has been previously used

as characteristic symptom to identify Eutypa disease but it can also be produced by Pm. viticola and

botryosphaeriaceous species. Therefore, the identification of the pathogens causing grapevine trunk diseases has relied on isolation of the pathogen from the symptomatic diseased samples onto artificial media and subsequent identification based on colony characteristic or anamorph morphology. This is a time-consuming process and requires destructive sampling of vines. Also these colony and

morphological characters are insufficient to identify some of the grapevine trunk pathogen species. For

example, the hyphae of E. lata have no unique diagnostic features which make the identification of

this species dependent on conidial characteristic. However, production of conidia from E. lata colonies

may take up to 2 months and some isolates do not produce conidia in cultures (Carter, 1991), making

et al., 2004). Similarly, the detection of another grapevine trunk pathogen, Pa. chlamydospora, from field samples also required destructive sampling for the isolation of fungi and identification. However, slow growth of the fungus in culture could delay the identification for several weeks (Pascoe & Cottral, 2000). Therefore, there is a requirement for a rapid, reliable and non-destructive sampling technique which would enable growers to identify the grapevine trunk pathogens even before the appearance of symptoms.

Recent advancement in molecular techniques can provide rapid, accurate and reliable detection of fungal pathogens. Since the introduction of PCR (Mullis & Faloona, 1987), nucleic acid base methods have been increasingly developed for the detection and identification of plant pathogens. The PCR is a procedure which allows amplification of a specific DNA sequence of the target species and is

considered a powerful technique for detecting species because of its specificity and sensitivity

(McCartney et al., 2003). The PCR-based detection methods are very sensitive and can be used to

detect very small quantities of pathogen DNA (Lee and Taylor, 1990). Therefore, the PCR based methods are useful for the identification of early stage or latent infections of plant pathogens. Development of species specific PCR assays for the detection of a species based on the unique DNA sequence of the target species is important. Different studies have shown that species specific PCR

assays developed for Pa. chlamydospora could detect this species in grapevines (Ridgway et al., 2002;

Retief et al., 2006). Similarly, species specific primers designed based on the ITS sequences for E.

lata was used to detect this species from infected grapevines (Lecomte et al., 2000). However, in some

cases combination of two different molecular techniques was required to identify the species. For

example, Rolshausen et al. (2004) developed a PCR-RFLP based protocol capable of identifying E.

lata following amplification of DNA using the universal ITS1 and ITS4 PCR primers and digestion of

the PCR product with the restriction enzyme AluI. However, in some assays the sensitivity of the

conventional PCR assay was sometimes inadequate to detect the target species especially from environmental samples. The nested-PCR method, which includes an extra step of amplification within

the target sequence, has been used to improve the sensitivity of detection (Whiteman et al., 2002).

Similar to other grapevine trunk pathogens, there are number of PCR based detection methods published for the detection of different botryosphaeriaceous species from different hosts.

1.8.1 Detection of botryosphaeriaceous species from field samples

There is a need for a rapid and effective tool to identify the botryosphaeriaceous species, especially in

a host infected by multiple species (Mohali et al., 2007). In prior research, different molecular markers

have been developed for PCR- based identification of some botryosphaeriaceous species (Zhou et al.,

2001; Burgess et al., 2003; Slipper et al., 2004a). Most of these molecular markers have been used for

botryosphaeriaceous species identification in laboratory conditions, using pure cultures. A quantitative PCR assay using species specific primers designed based on the sequence of the small subunit of

rRNA along with a TaqManTM probe has been used for the detection and quantification of Sphaeropsis

sapinea (D. pinea) from pine trees (Luchi et al., 2005). This quantitative PCR has been reported to

(2006) developed species specific PCR assays based on the mitochondrial small subunit ribosome

gene (mt SSU rDNA) for the detection of D. pinea and D. scrobiculata in dead red and jack pines in

which the pathogens caused collar rot. They designed species specific forward primers DpF and DsF for D. pinea and D. scrobiculata, respectively, and used the nonspecific reverse primer BotR to produce species specific detection. The resultant assay was useful to detect these species from the stem

or root of the diseased plant. Further to this, Luchi et al. (2011) recently developed a PCR method

using high resolution melting analysis (HRMA) for the detection of D. pinea in pine. This method

used the sequence of mitochondrial SSU rRNA to detect D. pinea and could distinguish D. pinea from

the closely related species D. scrobiculata without the need for a post-PCR procedure (Luchi et al.,

2011).

There are few reports available about the application of molecular markers for the detection of

botryosphaeriaceous species infecting grapevines. Recently, a nested PCR assay was developed for the

detection of botryosphaeriaceous species with particular emphasis on the N. parvum/ N.ribis complex

(Spagnolo et al., 2010). This method used the ITS5 and ITS4 (White et al., 1990) along with the

specific internal primers NprcA and NprcB designed for the N. parvum/ N. ribis complex and BoitsA

and BoitsB for botryosphaeriaceous species. This method was reported as specific and sensitive, being

able to detect these species in symptomatic and non-symptomatic vines (Spagnolo et al., 2010). In

addition to this, a PCR-SSCP assay was developed using ITS based multi genus primer pairs BOT100F and BOT472 which were able to differentiate four botryosphaeriaceous species found on grapevine including N. australe, N. luteum, D. mutila and D. seriata (Ridgway et al., 2011). The sensitivity of this assay was 0.1 pg in nested PCR assay and it was able to detect multiple

botryosphaeriaceous species in rainwater samples collected from the vineyards (Ridgway et al., 2011).

These methods are useful to screen the grapevine planting material for botryosphaeriaceous species infection.