Methods for Detection of Phytophthora fragariae var . rubi on Raspberry

Mirjana Koprivica1, Ivana Dulić-Marković2, Radivoje Jevtić3 and Dave E.L. Cooke4 1Ministry of Agriculture, Forestry and Water Management, Plant Protection Directorate, 11070 Belgrade, Omladinskih brigada 1, Serbia (mirjana.koprivica@minpolj.gov.rs) 2FAO SEEDEV 3Institute of Field and Vegetable Crops, 21000 Novi Sad, Maksima Gorkog 30, Serbia 4 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received: July 17, 2009 Accepted: September 10, 2009


INTRODUCTION
Raspberry root rot caused by species from the genus Phytophtora is known since 1937, but it was not considered an important disease of raspberries until mid-1980s.Several epiphytotics have been reported since then in central and eastern United States, Great Britain and Australia (Wilcox, 1997).It is considered that species of the genus Phytophtora are primary causes of raspberry decline in all production areas in the world (Figure 1).
Diagnostics of Phytophthora species is often based on a combination of microscopic examination of raspberry roots, isolation of selective media and exposure of susceptible plants ("bait plants") to infection by material under examination (Hughes et al., 2000).The first two methods are unreliable and the third requires 5-6 weeks of experiment and different bait plants for detection of different Phytophthora species.
A range of quick, sensitive and very specific diagnostic methods based on polimerase chain reaction have been developed owing to an extensive DNA database of several thousands isolates of Phytophthora, which are almost all species described (Bonants et al., 1997).ITS sequences (Internal Transcribed Spacers) present parts of genes that code ribosomal DNA (rDNA) and are variable between species.Simple extraction of DNA, followed by nested PCR, is a quick and reliable method for identification of Phytophthora species (Duncan, personal communication).Generic primers ITS 4 and DC 6 in the 1 st round of PCR amplify a part of ITS sequence of any Phytophtora including other Oomycetes in sample, while in the 2 nd round primers DC 1 and DC 5 amplify a product specific only for P. fragari ae."Universal" Phytophthora primers amplify ITS sequences of almost all Phytophthora and none other genus.Primers ITS 6, ITS 7, and ITS 8 are versions of the "universal" ITS primers ITS 5, ITS 2 and ITS 3, respectively, of White et al. (1990), modified by D.E.L. Cooke to improve the amplification of rDNA from Oomycetes.Restriction digestion with frequent cutter enzyme enables detection down to species level based on the number and size of bands in gel when compared with known species.This paper reports on detection of P. fragariae in raspberry samples showing root rot using isolation and molecular techniques.

Visual examination and sampling
A survey for P. fragariae var.rubi was conducted early in the spring, while plants were still low and patches of wilting or individual wilted plants in plantation were easy to observe (Figure 1).A number of plants were taken during sampling, which was not standardized by EPPO in that moment and depended on the sampler's personal judgement.
Raspberry samples with root rot symptoms were collected from several locations in western Serbia during the survey and are presented in Table 1.
During visual inspection, plants were examined for a reduction in the number of smaller roots, change of their colour from white to dark, and appearance of reddish discoloration after removal of bark on older root parts (Figure 2).

Isolation
Selective French Bean Agar (FBA, French bean -30 g, agar -15 g, water up to 1 l) was used for isolation from roots.After autoclaving and cooling near melting temperature FBA was amended with fungicides (bavistin, 0.6% of final concentration, and hymexasol, 0.6% of final concentration) and antibiotics (rifamycin, 0.3% of final concentration, and piramycin, 0.1% of fi- nal concentration).Small raspberry roots, 1.5-2.0cm in size, were sterilized in a 10% solution of NaOHCl, washed with sterile distilled water and, after drying on filter paper, put into media.Plates were incubated at 14ºC for 3-4 weeks.

Extraction of DNA
Small raspberry roots were washed under tap water, dryed on filter paper and ground in liquid nitrogen.An amount of 0.03 g of ground sample was placed in-to 1.5 ml eppendorf tubes, and an equivalent volume of insoluble polyvinilpirrolidon (PolyclarSB100) and 1 ml of extraction buffer (200 mM Tris HCl, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) were added to each.The tubes were placed onto a rotary shaker for 10 min, centrifuged at 10000 g for 5 min and 400 μl of supernatant was transferred to new 1.5 ml eppendorf tubes, then 400 μl of i-propanol was added and centrifuged again at 10000 g for 5 min.Supernatant was poured out and the pellet resuspended in 70% ethanol, and centrifuged at 10000 g for 5 min.The pellet was resuspended in 100 μl of sterile distilled water.
Mini spin columns (Thistle Scientific (Axygen) were filled up to 1 cm from top with polyvinilpirolidon (PVP), 250 μl of sterile distilled water was added and centrifuged at 4000 g for 5 min to allow collection of water at the bottom of tube.Columns were put in new eppendorf tubes with flat bottoms, 100 μl of extracted DNA was added and the tubes were centrifuged at 4000 g for 5 min.Pufiried DNA was stored at -20ºC.
Beside this method of purification, another one was also amended for raspberry samples by adding 500 μl of sterile distilled water instead of 100 μl after the extraction of DNA.In tubes with 500 μl of extracted DNA, 400 μl of 24:1 mixture of chlorophorm: isoamyl alcohol was added and centrifuged at 10000 g for 5 min; 400 μl of the upper liquid phase was pippeted into new eppendorf tubes and 200 μl of 5M NaCl, 600 μl i-propanol was added, and the tubes were put at -20ºC for 15  min to allow DNA to precipitate.The pellet was resuspended in 70% ethanol after centrifugation at 10000 g for 2 min and centrifuged again at 10000 g for 5 min.
Purified DNA was resuspended in 100 μl of sterile distilled water and stored at -20ºC before use.The success of DNA extraction was checked by running on 1% agarose gel for 30 min.

PCR amplification
DNA was amplified by nested PCR using ITS 4 and DC 6 set of primers in the first round and DC 1 and DC 5 in the second.Besides this one, extracted DNA was amplified by a conventional PCR using "universal" Phytophthora primers.
Master mix for the first and second rounds of PCR was the same containing: 19 μl of sterile distilled water, 0.5 μl of each forward and reverse primer (10 μM), 5 μl of extracted DNA from sample (10 times diluted) and 1 Ready to Go TM PCR bead (Amersham pharmacia biotech).The first round of PCR was performed in a thermal cycler under the following conditions: initial denaturation at 94ºC for 2 min, followed by 35 cycles of denaturation at 95ºC for 30 s, annealing at 56ºC for 30 s and elongation at 72ºC for 60 s and final extension at 72ºC for 10 min, while temperature was 66ºC in the second round of PCR annealing.
The products of nested PCR were visualized after running on 1% agarose gel (60V) for 30 min.The gel was stained in 0.05% solution of ethidium bromide.

Digestion of products amplified with "universal" Phytophthora primers
Msp1 enzyme was used for digestion of products amplified with "universal" Phytophthora primers.Master mix for digestion was: 10 μl of PCR products, 2 μl of 10 x enzyme buffer, 1 μl of Msp1 and 7 μl of sterile distilled water.Products were digested overnight at 37ºC.
Products were visualized by running on 2.5% NuSieve agarose gel (30V) for 3 hours and 30 min.The gel was stained with 0.05% solution of ethidium bromide.

Isolation
Only 2 of 200 potential isolates were obtained after 4 weeks.Colony was observed after 14 days of incubation (Figure 3).It was white coloured, mostly immersed in media and partly aerial.Nonseptate mycelia were observed during microscopic examination (40x) which got some septae as the culture became older (Figure 4).Sympodial branching was not observed.Individual sporangia were not observed directly in the culture, but they were observed when mycelial discs were exposed to soil solution and +4°C temperature, and zoospores were observed directly.This was an indication that some Phytophthora was isolated.

DNA extraction
Extraction from samples originated from the localities Košice and Krstac (samples 1 and 6) was not performed because of an insufficient quantity of roots, but it was successful from the other samples (Figure 5).

DNA amplification
The product specific only for Oomycetes, obtained after amplification with ITS 4 and DC 6 primers, was found only in samples originating from the locations Ostružanj (samples 4 and 5) and Vigošte (sample 10) (Figure 6).Althougth DNA was successfully extracted from all except samples 1 and 6 (Figure 5), prod-ucts from samples originating from Virovo, Košica, Pridvorica, Gruda and Viča were not observed.
In the second round, when DC 1 and DC 5 primers were used, 533 bp product specific for P. fragari ae was not only observed in samples from Ostružanj and Vigošte (product of 1310 bp observed in the first round) but also in samples from Pridvorica, Gruda and Viča (Figure 7).
After amplification with the "universal" Phytophthora primers, products were observed in samples from Ostružanj, Vigošte, Pridvorica, Gruda and Viča, but also in suspected samples (Ostružanj 2 and Gruda) in which the expected product was not detected by nested PCR (Figure 8).The "universal" Phytophthora primers provided better sensitivity and new bands were obverved (samples from Ostružanj 2 and Gruda) (Figure 8).

Digestion of products amplified with "universal" Phytophthora primers
After digestion with Msp1, bands specific for P. fra gariae were detected, which was confirmed by a corresponding number and size of bands, compared with the DNA extracted from a pure culture of P. fragari ae (Figure 9).None of the other Phytophthoras was detected in tested samples (Table 2).

DISCUSSION
Althought symptoms can be observed at almost any time of the year, the best time for visual inspection and sampling is springtime.In the summer, fungi are inactive and in the form of oospores because of higher temperatures.When the weather is cooler during sum- Table 2. Samples with or without root rot symptoms from different locations and results of the first and second round of PCR, conventional PCR using "universal" Phytophthora primers and digestion with Msp 1 mer fungi become active and can infect roots, but sampling needs to be very careful because advanced infection cannot be detected even by PCR test (Dave Cooke, personal communication).It is neccessary to take as many samples as possible, but the number has not yet been standardized.
Coinciding with the findings of Hughes et al. (2000), this examination showed that diagnostics of Phytophthoras based on a combination of microscope examination of small raspberry roots and isolation on selective media is unreliable and time consuming.This is very important for production of planting material because different Phytophthora can be detected that way, while only Phytophthora fragariae var.rubi is regulated for raspberries.
The most reliable PCR to be recommended is either nested PCR using DC 1 and DC 5 in the second round or conventional PCR using "universal" Phytophthora primers followed by digestion with restriction ensymes.

Figure 1 .
Figure 1.Wilting of raspberry plants in commercial plantations caused by Phytophthora fragariae var.rubi

Figure 2 .
Figure 2. Symptoms of raspberry root rot, small rootsreduction of number and change of colour from white to dark and apperance of reddish discoloration after removal of bark

Figure 3 .Figure 4 .
Figure 3. Phytophthora spp.culture Figure 6.Products amplified with ITS 4 and DC 6 primers in the first round of PCR

Figure 5 .
Figure 5. Extracted DNA from samples