Study of subpopulations of the chestnut blight
(Cryphonectria parasitica) fungus in the Carpathian basin
László Radócz
Department of Plant Protection, University of Deberecen, 138. Böszörményi str. H-4032, Hungary radocz@helios.date.hu
Abstract
During the last few decades destruction of European chestnut (Castanea sativa) populations caused by the chestnut blight fungus (Cryphonectria parasitica) has increased in Central Europe.
A total of 650 isolates (from 21 chestnut stands in southern and western parts of Hungary, three stands in Romania and six different sites in the Sub-Carpathion region of the Ukraine) were examined for virulence, classification of vegetative compatibility types (vc types) and for the presence of dsRNA.
Up to now 18 vc types have been found in Hungarian chestnut stands, but only one in Romania and one in the Sub-Carpathion region of the Ukraine. Long dsRNA fragments with similar electrophoretic characteristics were found in 36 Hungarian hypovirulent strains. These hypovirulent strains showed reduced virulence in vivo, as well as the in vitro(RADÓCZ1998a).
Keywords: biological control, hypovirulence, Endothia parasitica, vegetative compatibility types, dsRNA
1 Introduction
The blight fungus (Cryphonectria parasitica [Murr.] Barr) threatens chestnut stands of Central Europe including Austria (DONAUBAUER1964), Hungary (KÖRTVÉLY 1970), the Slovak Republic (JUHASOVA 1976), and Romania (FLOREA and POPA 1984). Some Austrian, Hungarian and Slovak chestnut stands have already been seriously damaged by this pathogen. Cryphonectria parasiticahas not, however, been reported so far in the Sub- Carpathian region of Ukraine. The main objectives of this research conducted under the framework of COST G4 were an extensive sampling and collection of C. parasiticaisolates in the Carpathian basin (Fig. 1), the determination of their vegetative compatibility (vc) types and the detection of the presence of the dsRNA. This information will help us to understand better how the disease spreads and to chose appropriate, local, hypovirulent fungal strains for sufficient biological control.
2 Materials and methods
Isolation and characterisation of fungal isolates: Bark samples were removed with a sterile cork borer from randomly selected cankers. Within 24 hours all the bark samples were surface sterilised in 70% ethanol, washed in sterile, distilled water and placed on PDA-medium (Difco) at 25 ºC.
After a few days small agar blocks with visible hyphae were removed and grown on PDAmb (ANAGNOSTAKIS 1977). One C. parasiticaisolate per canker was randomly selected for futher analysis. All isolates were stored on slants of PDAmb at 4 ºC in the dark.
Observations of C. parasiticaisolates morphology were made on PDAmb. The white cultural appearance of C. parasiticais correlated with the infection of the fungus by the Cryphonectria hypovirus (HEINIGERand RIGLING1994). Plates were inoculated with 5 mm plugs of mycelia removed from the margins of 7 day-old cultures with a sterilised cork borer. All isolates were
grown on PDAmb plates at 25 ºC in the dark for seven days, followed by incubation under day- light at room temperature for 7 days. Under these conditions, hypovirus-free isolates produced visible orange pigments, while hypovirus-infected ones remained white. Each white Hungarian isolate was tested for its ability to convert virulent “orange” strains as described by ANAGNOSTAKISand DAY(1979) during the course of in vitroconversion tests.
Test for vegetative compatibility type: The fungal strains were paired on PDA-Powell media (POWELL1995). Vegetative incom patibility and the mycelial death of C. parasiticawere indicated with a pH-indicator (bromocresol-green) in this type of media. Agar plugs containing mycelia were removed from the margins of seven-day-old PDA cultures and were paired 1 mm apart on the PDA-Powell media (1 cm from the edge of a 9-cm Petri dish) with photoperiods as described above.
The vegetative compatibility type was assessed according to the mycelial-barrage response using tester strains of EU 1-31 (CORTESIet al. 1998).
The Shannon diversity index (ROBINet al. 2000) was calculated for each population and sub- population for assessment and comparison of vc type diversity.
Detection of dsRNA: dsRNA was isolated from 7- to 10-day-old cultures as described by MORRIS and DODDS(1979). The presence of dsRNA on each isolate was assessed by agarose gel electro - phoresis. The dsRNA was electrophoresed using 1.2% agarose gels in 1xTBE at 80 Volts, stained with ethidium-bromide (0.25 mg/ml) and visualised by 300 nm UV-light. The isolates EP713 and EP155 were used as positive and negative controls in each test.
3 Results
Culture phenotype: The culture morphology of nearly 600 Hungarian isolates is listed in Table 1. Thirty-six isolates exhibited the white phenotype typical of European hypovirulent strains. These dsRNA-containing isolates were easily distinguished from dsRNA-free isolates by their colony morphology. The reduction in growth rate and the lack of aerial mycelium were typical in these Hungarian hypovirulent isolates.
Vegetative compatibility types: The vc type of each C. parasiticaisolate collected since 1993 was determined. The test showed at least 18 vc types occurring in Hungary, one in Romania and one in the Ukraine (Table 1). These 18 vc types were found in 30 plots. The four vc types EU-1, EU-6, EU-12 and EU-13 were dominant. A few vc types were represented by only one or two isolates, i.e. EU-3, EU-21, and EU-22. Five vc types, EU-2, EU-5, EU-6, EU-12, and EU-13, contained both orange and white isolates. All the Romanian and Ukrainian isolates were assigned to the vc type EU-12. No white isolates of C. parasitica have been found in these countries so far.
In seven Hungarian test sites: 1 (Ágfalva), 5 (Fertöszentmiklós), 11 (Rezi), 15 (Iharos - berény), 16 (Zengövárkony), 17 (Pécsvárad), and 19 (Gödöllö) perithecia were found, indi- cating that sexual reproduction of the fungus was taking place on these plots.
dsRNA detection: 36 of the Hungarian isolates with white colony morphology and reduced virulence (RADÓCZ1998b) were considered to be potentially hypovirulent. They all con- tained a L-dsRNA (approx. 12.7 kb) as demonstrated by agarose gel electrophoresis.
Table 1. Distribution of vegetative compatibility types of Cryphonectria parasiticain the Carpathian basin.
a the numbers of isolation plots correspond to Fig. 1. H = Hungary, RO = Romania, UR = Ukraine
b number of isolates. o: orange (virulent); w: white (hypovirulent)
c EU testers used EU 1-31. Testers that did not show any compatibility were omitted. + vegetative compatibility
d Shannon diversity index, H’= -∑pi ln pi, where pi is the frequency of the ithvc type
Sitesa No. of EU tester strainsc No. of H’d
isolatesb vc
types o w 1 2 3 4 5 6 9 11 12 13 14 15 16 17 21 22 28 29
1-Ágfalva (H) 45 - + + 2 0.19
2-Brenn-
bergbánya (H) 12 - + 1 -
3-Bánfalva (H) 15 - + 1 -
4-Fáber rét (H) 16 - + 1 -
5-Fertöszent-
miklós (H) 46 - + + 2 0.35
6-Cák (H) 37 5 + + 2 0.33
7-Csepreg (H) 35 - + + 2 0.27
8-Csipkerek (H) 28 - + 1 -
9-Velem (H) 31 6 + 1 -
10-Szombathely 9 - + 1 -
11-Rezi (H) 56 15 + + 2 0.46
12-Nemeshetés 14 - + + 2 0.42
13-Zalaegerszeg 16 - + 1 -
14-Sand (H) 20 4 + + 2 0.31
15-Iharosberény 45 6 + + + 3 0.68
16-Zengövárkony 65 - + + + + + 5 0.98
17-Pécsvárad (H) 36 - + + + 3 0.73
18-Pécs (H) 23 - + + 2 0.33
19-Gödöllö(H) 13 - + + 2 0.48
20-Nagymaros 16 - + + 2 0.22
21-Budapest (H) 3 - + 1 -
22-Baia Mare I
(RO) 6 - + 1 -
23-Baia Mare II
(RO) 9 - + 1 -
24-Baia Mare III
(RO) 5 - + 1 -
25-Uzhgorod
(UR) 3 - + 1 -
26-Seredne (UR) 9 - + 1 -
27-Gluboka (UR) 6 - + 1 -
28-Bobovische I
(UR) 11 - + 1 -
29-Bobovische II
(UR) 15 - + 1 -
30-Gajdos (UR) 8 - + 1 -
213 4 7 6
98 10 13
20
15 1411
21 19
2526 22 2728
29 2324
1716 18 21
4 7
20
1411
21 19
1716 18
2526
22 2728
29 2324
Fig. 1. Sampled chestnut (Castanea sativa)populations in the Carpathian basin.
4 Discussion
This study represents the first examination of the Romanian and Ukrainian subpopulations of the chestnut blight fungus. In contrast to the Hungarian populations these nine subpopu- lations of the chestnut blight fungus were very uniform with only one vc type (EU-12) and no hypovirulent strains found. The vc type EU-12 occurs at several sites in Hungary includ- ing site 19 (Gödöllö), which is the closest to the Romanian and Ukrainian sites. Therefore it might be that the pathogen entered into these countries from Hungary.
In the 21 Hungarian study sites, a total of 18 vc types were identified. Most of the sites, however, contained only one or two vc types. Hypovirulent strains of C. parasitica(carrying dsRNA) were found at five sites, all in the west of the country, where many healing cankers indicate the disease situation. During the course of our six-year study, we found an increase in vegetative compatibility diversity and in the total number of vc types (RADÓCZ1995).
Only one vc type was found in plots 1, 6, 7, 12, 14, 15, 16, 17, 18 and 20 by the end of the year 1994. A very marked increase in the number of the detected vc types have been reported since that year, especially on the plots situated in the southern part of Hungary (16- Zengövárkony, 17-Pécsvárad, and 18-Pécs). This might be because of sexual recombination among local vc types. Vc types EU-1, EU-6, EU-12, and EU-13 are the most frequent ones in the Carpathian basin. They also represent the most widespread types in the test sites. It may, therefore, be that isolates belonging to vc types EU-6, EU-12 and EU-13 were the first to colonise Hungarian chestnut stands.
ELLISTON(1985) noted that hypovirulence-associated phenotypes exhibited by C. para- sitica isolates are mostly dependent on dsRNA rather than on the genotype of the host fungus. Hungarian hypovirulent strains showed homogeneity in the electrophoretic charac-
teristics of their dsRNAs and similarity in their effects on the phenotype of the host fungus (RADÓCZ 1998b). Studies by ALLEMANN et al. (1999) demonstrate that three CHV1 (Cryphonectria hypovirus) from Hungary all belonged to the Italian subtype.
To obtain more accurate results in describing the dynamics of C. parasitica, a cross-border collaboration will be necessary. Biological control of the C. parasiticafungus with hypoviru- lent strains seems to be promising due to the low diversity of vc types found at the studied sites. This may provide a basis for a programme to restore European chestnut trees suffering from blight in the Carpathian basin.
Acknowledgements
Many thanks to Sandra Anagnostakis, Bradley Hillman, Ursula Heiniger, Daniel Rigling, Paolo Cortesi, Gabriela Juhasova and Eva Wilhelm for their help and for providing C. parasiticaisolates for our tests. Furthermore we thank Floarea Dumitrescu and Vasyl Chrochko for their research collaboration and for the research fellowship from the Bólyai Foundation of the Hungarian Academy of Sciences.
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Accepted 28.1.02
