GDK 176.1 Castanea sativaMi\\.+A\\.\6 Cryphonectriaparasitica:(439) Prispelo / Received: 19.03.2002	Izvirni strokovni članek
Sprejeto / Accepted: 06.06.2002	Original professional paper
distribution of chestnut blight in hungary and characterization of the hungarian chestnut blight {CRYPHONECTRIA PARASITICA (murill) barr) strains
Ilona APONYI*, Gizella IZSÄNYl", Jänos SÖTONYl", Krisztina VARGA*, Zsolt VENDREI*
Abstract
During surveys made in the main sweet chestnut growing regions in Hungary we observed that one of the most serious "adversaries" of sweet chestnut trees is chestnut blight caused by Cryphonectria parasitica. Our main goals were to survey the plant health status of the Hungarian sweet chestnut stands, with special attention to chestnut blight, and to implement a biological control method against chestnut blight based on hypovirulent strains of C. parasitica. The virulence of the strains was characterised by different laboratory tests: morphological examination, virulence test, phenol oxidase test, dsRNA extraction, VCG test and artificial inoculation were performed on experimental plots.
Key words: biological control, Cryphonectria parasitica, Hungary, hypovirulence, Castanea saliva, forest health status, Hungary
RAZŠIRJENOST IN KARAKTERIZACIJA SEVOV KOSTANJEVEGA RAKA (CRYPHONECTRIA PARASITICA (MURILI) BARR) NA MADŽARSKEM
Izvleček
Med popisi, ki smo jih opravili v območjih razširjenosti pravega kostanja na Madžarskem, smo ugotovili, daje kostanjev rak (povzroča ga Cryphonectria parasitica) glavni "sovražnik" kostanjevih dreves. Cilja popisa sta bila: (a) ugotoviti zdravstveno stanje dreves pravega kostanja na Madžarskem (s poudarkom na kostanjevem raku): (b) vpeljati biološko kontrolo kostanjevega raka z uporabo hipovirulentnih sevov C. parasitica. Virulenco sevov smo določili z različnimi laboratorijskimi testi: analizo morfoloških lastnosti, testom virulence, fenol oksidaznim testom, ekstrakcijo dsRNA, testom vegetativne kompatibilnosti in umetno inokulacijo na poskusnih ploskvah.
Ključne besede: biološka kontrola, Cryphonectria parasitica,, hipovirulenca, Castanea sativa, zdravstveno stanje gozda. Madžarska
' Central Service for Plant Protection and Soil Conservation, 1118 Budapest, Budaörsi üt 141-145, Hungary " Plant Protection and Soil Conservation Service of Zala County, 8900 Zalaegerszeg, Kinizsi lit 81, Hungary
contents
VSEBINA
1	introduction
UVOD...............................................................................................63
2	material and methods
MATERIAL IN METODE...............................................................65
3	results
REZULTATI.....................................................................................72
4	discussion
RAZPRAVA.....................................................................................74
5	povzetek....................................................................................75
6	references
VIRI..................................................................................................76
1 introduction
UVOD
1.1 ECONOMIC IMPORTANCE OF SWEET CHESTNUT GOSPODARSKI POMEN PRAVEGA KOSTANJA
European sweet chestnut is a tree native to the Carpathian basin. It is a natural and valuable tree species in Hungary, known through the centuries as a forest tree. There are no large continuous sweet chestnut stands; the largest one is at Zengövärkony, Baranya County (38 hectares). Unfortunately, a lot of stands are abandoned and visited only at harvesting time. However, there are also well-managed stands.
The most important stands are situated in the western part of the country, in the counties of Somogy, Vas, Veszprem and Zala, as well as in the Danube bend (Figure 1).
Figure 1: Hungarian sweet chestnut growing regions
Slika 1: Območja razširjenosti pravega kostanja na Madžarskem
The sweet chestnut area is not significant: approximately 200.000 ha. The decrease has mainly been caused by Cryphonectria parasitica, which is responsible for chestnut blight. As the quality of trees is not suitable for timber production, the trees are mostly used for fruit and tannin extraction.
The utilization of sweet chestnut is as follows:
orchards: a highly nutritive food rich in vitamins, forestry trees: in the ship, furniture and upholstery industries, one of the most beautiful ornamental trees, honey-bearing and pharmaceutical raw materials, tannin extraction.
1.2 ECONOMIC IMPACT OF THE CHESTNUT BLIGHT
GOSPODARSKI POMEN KOSTANJEVEGA RAKA
C. parasitica caused almost complete destruction of Castanea dentata in America between 1904 and 1950. In Europe, it has been extensively spreading from Italy on C. saliva since 1938. The pathogen was first found in Hungary in 1969 at Nemeshetes, Zala county (KÖRTVELY 1970).
During surveys, C. parasitica was detected in all significant chestnut groves, orchards and in several plantations producing propagation material. As a result, in 1972 C. parasitica was declared a quarantined pest. In the 1970s, 1.100 ha of sweet chestnut orchards were planted in the western part of the country. These stands have almost completely disappeared due to planting deficiencies, frost damage and chestnut blight. Natural and scattered stands are also infected and degraded.
According to surveys conducted during the past few years, the presence of hypovirulent strains was observed in every sweet chestnut growing region in the country except the Danube bend.
1.3 BIOLOGICAL CONTROL OF CHESTNUT BLIGHT BIOLOŠKI NADZOR KOSTANJEVEGA RAKA
Lacking an effective pest management programme, biological control seems to be one possible solution against chestnut blight. This method is based on using the hypovirulent strains of C. parasitica that are the cause of superficial canker. The virulence of these strains is low due to the dsRNA mycovirus in the cytoplasm (SHAPIRA et al. 1991). Trees infected by hypovirulent strains can survive the infection by callus formation.
Hypovirulence can be transmitted by hyphal anastomosis from hypovirulent strains to virulent ones of the same vegetative compatibility group. The virulent strains can be altered by the hypovirulent ones from the same vegetative compatibility group through hyphal anastomosis. Thus, the virulent strains lose their highly pathogenic character and become in their turn "hypovirulent".
In stands where the hypovirulent pressure is higher than the virulent one, the problem is solved naturally in a suitable silvicultural management system. Prunings and cleanings in chestnut fruit orchards are necessary.
Our purpose is to artificially spread hypovirulence where it is not naturally present or its ratio is low.
2 material and methods
MATERIAL IN METODE
We conducted surveys in sixteen plots located in five counties: Zala, Vas, Veszprem, Baranya and Pest.
2.1 FIELD EXPERIMENTS
TERENSKI POSKUSI 2.1.1 Selection of the experimental plots
Izbor poskusnih ploskev
Sixteen experimental plots were established in the main sweet chestnut growing regions in the western part of the country (Baranya, Vas, Veszprem and Zala counties) and in the Danube bend region (Pest county).
The types of experimental plots are different: natural sweet chestnut stand, mixed forest, scattered plot and orchard (cultivated and abandoned) (Table 1).
Table 1:	Hungarian sweet chestnut experimental plots
Preglednica 1: Poskusne ploskve pravega kostanja na Madžarskem
Place Kraj	Area Površina (ha)	Type of the stand Seslojni tip	Natural hipovirulence Naravna hipovirulentnost
Zala countv			
Nagykutas	5	Planted orchard / Nasad	Present / Prisotna
Nagylengyel	1	Planted orchard / Nasad	Present / Prisotna
Nemeshetes	4	Mixed forest / Mešan gozd	Present / Prisotna
Palin	16	Planted orchard / Nasad	Present / Prisotna
Pätro	2	Planted orchard / Nasad	Present / Prisotna
Mihäld	2	Scattered / Posamezno drevje	Present / Prisotna
Zalamerenye	10	Mixed forest / Mešan gozd	Present / Prisotna
Zaiaüjlak	20	Mixed forest / Mešan gozd	Present / Prisotna
Rezi	30	Natural sweet chestnut Naravni pravi koslani	Present / Prisotna
Vas countv			
Csepreg	36	Mixed forest / Mešan gozd	Present / Prisotna
Velem	27	Scattered / Posamezno drevje	Present / Prisotna
Csipkerek	23	Planted orchard / Nasad	Present / Prisotna
VeszDrim countv			
Veszprem	0,1	Planted orchard / Nasad	Present / Prisotna
Tds	0,1	Planted orchard / Nasad	Present / Prisotna
Baranya county			
Zengövärkony	38	Natural sweet chestnut Naravni pravi kostanj	Present / Prisotna
Pest countv			
Nagymaros	14	Natural sweet chestnut Naravni pravi kostanj	Not present / Ni prisotna
2.1.2 Evaluation of plant health status
Ocena zdravstvenega stanja dreves
We evaluated plant health status with special attention to ink disease and chestnut blight. We assessed the different types of blight infection: old damage or new infection and the percentage of the new, possibly hypovirulent infections was determined. Fifteen to fifty trees were evaluated by morphological observation per each plot. We focused mainly for the new hypovirulent infection.
2.1.3 Characterization of the different types of chestnut blight infection
Karakterizacija različnih tipov infekcij s kostanjevim rakom
Ali kinds of cankers were determined according to groups such as: abnormal (hypovirulent), intermediate and normal (virulent) canker. Detecting the percentage of abnormal canker is very difficult and not reliable in the field, therefore laboratory tests were performed.
C. parasitica is a wound parasite and can cause infection on the shoots, branches and bark of Castanea sativa. There are three different strains of the fiingus: virulent, intermediate and hypovirulent. Virulent strains can cause serious sunken cankers. Cankers may enlarge so rapidly that the stem becomes girdled without callus formation. Typical symptoms of early infection on young shoots are: reddish, sunken bark, cracks, formation of epicormic shoots, ftingal stroma production.
Hypovirulent strains are responsible for superficial infection. Callusing may occur only as a healing phenomenon.
A typical characteristic of the infection is a mycelial fan of C. parasitica in the inner bark of the chestnut tree. In humid weather, masses of yellow-orange to reddish-brown pustules develop on the infected bark and exude long orange tendrils of spores.
2.1.4 Sampling of C. parasitica strains from different types of cankered wounds
Vzorčenje sevov C. parasitica iz različnih tipov rakastih ran
We collected more than 300 samples during the surveys from each type of cankered wound; about 15 samples were collected per each plot. Samples approximately 2 x 2 cm were taken from the bark, from the margin of the infected and healthy part of the plant tissue by a disinfected knife or scalpel. We focused mainly on the new hypovirulent infections.
Artificial inoculation: After the laboratory experiment, artificial inoculations were performed with selected hypovirulent strains in two experimental plots; in the western part of the country, Zala county, where natural hypovirulence is present, and in the Danube bend, which is the only region where the natural hypovirulent strains are not present. The main reason for this work was to artificially spread hypovirulence at the stand level.
Holes (with a diameter of 8 mm) were made in the bark tissue by electric drills in four sets, with four different hypovirulent strains, 6 cm far from each other on the same tree. Five day-old mycelia of the hypovirulent strain from PDA were put in the wound and the surface was closed with transparent adhesive tape to prevent rapid drying.
At first, we used two differently compatible hypovirulent strains in the plots (instead of four), according to Turchetti's method (TURCHETTI / MARESSI 1991). The inoculation was done while the chestnut trees were flowering. Mycelium of hypovirulent strains was introduced into the holes at the margin of infections.
2.2 LABORATORY EXPERIMENTS
LABORATORIJSKI POSKUSI
2.2.1 Isolation of C. parasitica from infected samples
Izolacija C. parasitica iz okuženih vzorcev
5x5 mm sections were cut out from the collected samples on the day after sampling and were placed on difco potato dextrose agar containing 100 mg/1 methionine, 1 mg/1 biotin (PDAmb) after disinfection by 96% ethanol and flaming. Culturing was done in the dark at 28°C.
2.2.2 Maintenance of the isolates
Vzdrževanje izolatov
Isolates were maintained on slant potato dextrose agar (PDA difco) without methionin and biotin under sterile paraffin oil at 4°C and in four replicates.
2.2.3 Characterization of the strains based on morphology
Karakterizacija sevov na osnovi morfologije
Determination based on the colour of the colony and the quantity of the fruiting bodies is not reliable. The virulent isolates are mainly reddish, yellow and the hypovirulent isolates have little or no pigment. These grow more slowly than normal virulent strains at 28°C on PDA media in the laboratory. However, there are many exceptions, therefore other tests are required.
2.2.4 Virulence test
Test virulence
Two replicates of 100 isolates were used in this experiment. After artificial inoculation, shoots (length: 30 cm, diameter: 1,5-2 cm) were put in the incubation tubes for two
weeks at 28°C under conditions of high relative humidity until evaluation. During the evaluation, the diameter of the infected part was measured and pycnidia production was observed.
2.2.5 Bavendamm (phenol oxidase) test
Bavendammov (fenol oksidazni) test
To test for phenol oxidase activity (RIGLING / HEINIGER / HOHL 1989), mycelia were cut out from the five to six days old colonies on potato dextrose agar and were placed on malt extract agar. It contains 0,5% tannic acid, 1,5% Difco malt extract, and 2% Difco bacto-agar, adjusted with NaOH to pH 4,5. After a few days of incubation at room temperature in the dark, all virulent strains produced a strong colour reaction, indicating phenol oxidase activity, but the hypovirulent strains showed weak or no reaction in the lack of or blocked by this enzyme. Over three years, 100 isolates were tested. Evaluation was based on positive control isolates (Table 2).
Table 2:	Control isolates used for Bavendamm test
Preglednica 2: Kontrolni izolati, uporabljeni za Bavendammov test
Laboratory code Laboratorijska oznaka	Origin Izvor	dsRNA extraction dsRNA ekstrakcija	Bavendamm test Bavendammov test	Virulence Virulenca
NI HV/1	Nagylengyel	+	-	weak hypovirulent šibko hipovirulenten
HLHV/4	Nagylengyel	+	--	hypovirulent hipovirulenten
NlHV/2	Nagylengyel	+	—	strong hypovirulent močno hipovirulenten
NpV/3	Nemespatro	-	+	weak virulent šibko virulenten
NgV/4	Nagygerind	-	++	virulent / virulenten
NmV/1	Nagymaros	-	+++	strong virulent močno virulenten
2.2.6 ds RNA extraction
dsRNA ekstrakcija
ds RNA extraction was performed according to MORRIS / DODDS (1979) method, but was later modified to make the extraction faster by excluding DNA digestion, because all of the DNA was eliminated during the test.
The strains were grown on PDAmb. Mycelia were harvested from 7-10 day-old colonies. Ig of fresh mycelia was homogenised in 2 x STE buffer (10 x STE: lOOmM NaCl, 50mM Tris, ImM EDTA) with 10% SDS, 0,5% bentonite. Incubation took place for 5 min. at room temperature. Before shaking for 40 min., at 300 rpm, room temperature, 6 ml water-saturated phenol and 6 ml of chloroform were added. After centrifugation, the ethanol content of the supernatant was adjusted to 17 % and loaded to the freshly prepared CF11 (Whatman) column. The column was washed through with 50 ml IxSTE-17% ethanol and at the end, the RNA was harvested in 1 x STE buffer. After precipitation, the RNA was separated by agarose gel electrophoresis in 1% agarose gel containing ethidium bromide, at 90V for 40 min. and evaluated under UV light.
2.2.7 Determination of vegetative compatibility (V-C) groups
Določitev vegetativno kompatibilnih (V-C) skupin
The importance of this test is to determine the vegetative compatibility (V-C) properties between the isolates based on the presence of hyphal anastomosis. The isolates were placed on potato dextrose agar at a 1-1,5 cm distance from each other. The test was evaluated after one week of incubation in light at 28°C. The dsRNA is transmitted through hyphal anastomoses and rapidly converts the virulent strain if the virulent strain is compatible to the hypovirulent one. In cases of incompatibility, barrage lines with many pycnidia develop between the strains.
3 RESULTS
rezultati
We established and conducted surveys in the main sweet chestnut growing areas of the country; in sixteen plots located in five counties in the western part of the country and in the Danube bend. Among the stands there were plantations producing propagation material, planted groves, coppices, 'natural' (more than 100 years old trees) sweet chestnut trees and mixed forests.
Unfortunately, many stands were abandoned and visited only at harvesting time but there were well-managed stands, too.
During the surveys, we evaluated the plant health status of these stands. We confirmed that in Hungary chestnut blight is one of sweet chestnut's main problems. Natural presence of the hypovirulent strains was observed in all sweet chestnut growing areas except one region (Table 3).
Table 3:	Shares of the hypovirulent, virulent and intermediate infections in the
different counties
Preglednica 3: Deleži hipovirulentnih, virulentnih in vmesnih infekcij v posameznih okrajih
Site	Hypovirulent (%)	Virulent (%)	Intermediate (%)
Ploskev	Hipovirulentni (%)	Virulentni (%)	Vmesni (%)
Baranya county	20	60	20
Pest county	-	85	15
Vas county	18	53	29
Veszprem county	25	40	35
Zala county	28	15	57
Ink disease was not found, probably due to the extremely dry weather of the last few years. Other foliage diseases, such as Mycosphaerella maculiformis, were not significant at the time of the surveys (flowering and fruit development period).
For laboratory examinations, we collected about 300 samples infected with C. parasitica during the three-year work. Samples from each type of cankered wounds were collected but we focused mainly on the new hypovirulent infections. Isolation of the fungus is not
difficult, only 10 % of the samples were lost during the isolation. The first stage of isolate observation was based on morphological properties, but as the morphological character is not reliable in itself, other tests were required.
Virulence testing was carried out on 50 isolates to observe the virulence of the isolates but this test was not suitable for hypovirulence selection.
Bavendamm (phenol oxidase) testing was performed using 100 isolates. Clear differences in phenol oxidase activity were found between virulent and hypovimlent strains of C. parasitica. This enzyme, responsible for the colour reaction, was identified as phenol oxidase of the laccase type. Laccase activity has been suggested of being involved in degradation of lignin, in pathogenesis, in formation of fruiting bodies and in pigmentation (RIGLING / HEINIGER / HOHL 1989).
Mainly morphologically positive, seemingly hypoviralent isolates were tested by the extraction of the 12.000 base pairs long double-stranded RNA (dsRNA) mycovirus (Figure 2). Strong correlation was observed between the results of phenol oxidase test and dsRNA extraction.
21.000 bp 12.000bp
1.700 bp 600 bp
Figure 2: Purified mycovirus (12.000 bp in size and sub-units: 1.700 bp and 600 bp) on
1% agarose gel (Marker: NBL GENE SCIENCE EcoRI/Hind III) Slika 2: Prečiščeni mikovirusi (velikosti 12.000 bp; podenote velikosti 1.700 bp in 600 bp) na 1 % agarju (Marker: NBL GENE SCIENCE EcoRI/Hind III)
Vegetative Compatibility Groups (V-CG) were determined within the plots and the proper hypovirulent strains were also selected for artificial inoculations.
Finally artificial inoculation was performed with the proper hypovirulent strains in two experimental plots: in the western part of the country, in Zala county where natural hypovirulence is present and in the Danube bend which is the only region where the natural hypovirulent strains are not present.
For reference, we have an experimental plot at Nagylengyel, county Zala, Twenty-seven trees out of 90 were inoculated with two different hypovirulent strains. The healing process was observed in this plot, but it is slow due to the high virulent pressure.
In the Danube bend, nearly 300 trees were inoculated with four different hypovirulent strains in the last year during sweet chestnut flowering.
4 discussion
RAZPRAVA
Studies of sweet chestnut trees have been performed by the Hungarian Plant Protection Service since 1993. In the framework of INCO-Copemicus project "CHESUD", we could continue this work with the implementation of the biological control method against chestnut blight.
We collected more than 300 samples during surveys from each type of cankered wounds for laboratory examination.
The virulence of the strains was characterised by different laboratory tests; morphological examination, virulence test, phenol oxidase test, dsRNA extraction and VCG test.
Determination based on the colour of the colony and the quantity of the fruiting bodies is not reliable and several studies suggest that no clear relationship exists between the white cultural phenotype and hypovirulence.
After the laboratory experiment, artificial inoculations were performed with the selected hypovirulent strains in two experimental plots: in the western part of the country, Zala county where natural hypovirulence is present and in the Danube bend which is the only region where the natural hypovirulent strains are not present. The main reason for this work was the artificial spread of the hypovirulence at the stand level.
Inoculations of active cankers with hypovirulent strains healed the cankers when strains with the appropriate V-C groups were used.
Biological control of chestnut blight in Hungary is very promising. Natural hypovirulence is present in almost every sweet chestnut stand. In the seventies 1.100 hectares of sweet chestnut orchards were planted in the western part of the country. These stands have been almost totally destroyed due to planting deficiencies, frost damage and chestnut blight. Sweet chestnut grove owners plan to establish new plantations and are hoping for a successful biological control method.
5 povzetek
Med popisi, ki smo jih opravili v območjih razširjenosti pravega kostanja v zahodnem delu Madžarske (okraji Baranya, Vas, Veszprem in Zala) ter v Podonavju, smo ugotovili, da kostanjev rak (povzroča ga Cryphonectria parasitica) resno ogroža kostanjeva drevesa. V popis smo vključili različne tipe ploskev: naravni sestoji pravega kostanja, mešan gozd, nasadi (kultivirani in opuščeni).
Cilja popisa sta bila: (a) ugotoviti zdravstveno stanje dreves pravega kostanja na Madžarskem (s poudarkom na kostanjevem raku); (b) vpeljati biološko kontrolo kostanjevega raka z uporabo hipovirulentnih sevov C. parasitica.
Prisotnost naravnih hipovirulentnih sevov smo ugotovili na vseh popisanih lokacijah, razen v Podonavju.
Za karakterizacijo reprezentativnega števila izolatov s poskusnih ploskev smo izvedli pet različnih laboratorijskih testov. Med popisi smo nabrali več kot 300 vzorcev vseh tipov
rakastih ran (po 15 vzorcev na ploskev), z namenom, da bi umetno vnesli hipovirulenco na ploskve, kjer le-ta po naravi ni prisotna oziroma je njen delež majhen.
S testom vegetativno kompatibilnih skupin (VGC test) smo na ploskvah določili vegetativno kompatibilne skupine in izbrali hipovirulentne seve, primerne za umetno inokulacijo.
Po laboratorijskih poskusih smo izvedli umetno inokulacijo izbranih hipovirulentnih sevov na dveh poskusnih ploskvah: (a) na zahodnem delu okraja Zala, kjer je prisotna naravna hipovirulenca; (b) v Podonavju, kije izmed popisanih območij edino, kjer nismo našli naravnih hipovirulentnih sevov. Namen inokulacije je bila umetna razširitev hipovirulentnih sevov v sestoju. Inokulacija s hipovimlentnimi sevi je uspešno zavrla razvoj raka v primerih, ko smo uporabili ustrezne vegetativno kompatibilne (V-C) skupine.
6 references VIRI
KÖRTVELY, A., 1970. A gesztenye endotiäs keregelhaläsa. (Bark Destruction Caused by Endothia parasitica (Murr) Anderson, on Chestnut Trees).- Növenyvedelem 6: 358-361.
MORRIS, T. J. / DODDS, J. A. 1979. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue.- Phytopathology 69: 854-858.
RIGLING, D. / HEINIGER, U. /HOHL, H. R., 1989. Reduction of laccase activity in dsRNA-Containing Hypovirulent strains of Cryphonectria (Endothia) parasitica.-Phytopathology 79: 219-223.
SHAPIRA, R. / CHOI, G. H. / HILLMAN, B. I. / NUSS, D. L., 1991. The Contribution of Defective RNAs to the Complexity of Viral-Encoded Double-Stranded RNA Populations Present in Hypovirulent Strains of the Chestnut Blight Fungus Cryphonectria parasitica.- EMBO Journal 10, 4: 741-746.
TURCHETTI, T. / MARESSI, G., 1991. Inoculation Trials with Hypovirulent Strains of Cryphonectria papasitica (Murr.) Barr.- Eur. J. Path. 21: 65-70.