MONITORING OF SWEET CHESTNUT BLIGHT ON PERMANENT PLOTS

U radu su prikazani rezultati analize praćenja zdravstvenog stanja pitomog kestena na trajnim pokusnim plohama, osnovanim na područjima uprava šuma podružnica Zagreb i Sisak. Osnovano je pet trajnih pokusnih ploha: Sljeme, Markuševac, Bistra, Vučjak Tješnjak i Šamarica. Motrenje raka kore obavlja se od 2002. godine. Rezultati pokazuju povećanje broja zaraženih i suhih stabala, kao i broja rakastih tvorevina u odnosu na broj zaraženih stabala te ukupan broj stabala iz čega se može zaključiti kako je rak kore pitomog kestena u progresiji. Razvoj bolesti potenciran je i sveukupnim lošim stanjem kestenovih sastojina.The paper presents the results of an analysis of monitoring the health status of Sweet chestnut on permanent experimental plots, established in the areas of the Forest Administration Branch Offices of Zagreb and Sisak. Five permanent experimental plots were established: Sljeme, Marku{evac, Bistra, Vu~jak Tje{njak and [amarica. Monitoring of blight/canker has been carried out since 2002. The results show an increased number of infected and dry trees, and the number of canerous formations in relation to the number of infected trees and the total number of trees, from which it can be concluded that Sweet chestnut blight/canker is in progression. Development of the disease is also potentiated by the overall poor condition of the chestnut stands

DNA Barcoding of Fungi in the Forest Ecosystem of the Psunj and Papuk Mountains in Croatia

The saprotrophic, endophytic, and parasitic fungi were detected from the samples collected in the forest of the management unit East Psunj and Papuk Nature Park in Croatia. The disease symptoms, the morphology of fruiting bodies and fungal culture, and DNA barcoding were combined for determining the fungi at the genus or species level. DNA barcoding is a standardized and automated identification of species based on recognition of highly variable DNA sequences. DNA barcoding has a wide application in the diagnostic purpose of fungi in biological specimens. DNA samples for DNA barcoding were isolated from infected tree tissues, fungal fruiting bodies or fungal cultures. The ITS or ITS2 sequences of the fungal DNA were sequenced and aligned with the reference sequences in GenBank (NCBI) using BLAST. The size of ITS and ITS2 sequences were 512-584 bp and 248-326 bp, respectively. The sequences showed a high identity of 97.21%-100% at 98%-100% coverage with reference sequences in GenBank (NCBI). The exception was the species Amphilogia gyrosa that showed 95.65% identity at 100% coverage. Two fungi were determined at genus level: Cladosporium sp., and Cytospora sp., while 11 fungi were determined at species level: Alternaria alternata, Aureobasidium pullulans, Amphilogia gyrosa, Capronia pilosella, Cryphonectria parasitica, Exidia glandulosa, Epicoccum nigrum, Penicillium glabrum, Pezicula carpinea, Rosellinia corticium, and Stereum hirsutum

Qualitative Properties and Mycoflora of Fresh and Stored Fruits of Sweet Chestnut (Castanea sativa Mill.)

Plod pitomog kestena (Castanea sativa Mill.) se najčešće upotrebljava za proizvodnju šumskog reprodukcijskog materijala za pošumljavanje i obnovu šuma, te kao prehrambeni proizvod. Lako je kvarljv i teško ga je uskladištiti bez značajnog gubitka vitaliteta. Cilj istraživanja je bio određivanje kvalitativnih svojstava i zdravstvenog stanja plodova pitomog kestena te detekcija vrsta gljiva i kukaca prije i tijekom skladištenja. Plodovi su sakupljeni u listopadu 2007. godine na području UŠP Požega. Analizirana su kvalitativna svojstva plodova prema standardnim ISTA metodama. Klijavost je iznosila 40 %, a vitalitet je iznosio 35 %. Uskladišten je u hladnjači HŠI na temperaturi 3 do 5 °C u PE vrećama. Nakon pet mjeseci skladištenja analizirano je zdravstveno stanje plodova. Analizom je utvrđeno visoko učešće zaraženih plodova u iznosu od 60 %. Gljive su determinirane klasičnom morfološkom analizom primjenom makro i mikro metoda i molekularnom metodom. Detektirano je osam vrsta patogenih gljiva i dvije vrste kukaca: Alternaria alternata (Fr.) Kisler, Aspergillus flavus Link., Aspergillus niger Tiegh, Ciboria batschiana (Zopf) Buchw., Penicillium spp., Phomopsis castanea (Sacc.) Höhn, Trichoderma citrinoviride Pers., Trichothecium roseum Link., i Curculio elephas Gill., Cydia splendana Hb.The fruit of the sweet chestnut (Castanea sativa Mill.) is most often used for the production of forest reproductive material for afforestation and reforestation, and as a food product. It is easily perishable and difficult to store without significant loss of viability.The aim of the study was to determine the qualitative properties and health status of sweet chestnut fruits and the detection of fungal and insect species before and during storage. The fruits were collected in the Forest Administration Požega in October 2007. The qualitative properties of the fruits according to ISTA Rules were analyzed. The germination was 40% and viability was 35%. The fruits were stored in PE bags in the refrigerator at a temperature of 3 to 5° C. After five months of storage, the health status of the fruits was analyzed. The analysis revealed a high percentage of infected fruits (60%) The fungi were determined by classical morphological analysis using macro and micro methods and molecular method. Eight species of pathogenic fungi and two species of insects were detected: Alternaria alternata (Fr.) Kisler, Aspergillus flavus Link., Aspergillus niger Tiegh, Ciboria batschiana (Zopf) Buchw., Penicillium spp., Phomopsis castanea (Saccichodide) Höhn, Tr. Pers., Trichothecium roseum Link., and Curculio elephas Gill., and Cydia splendana Hb

Qualitative Properties of Penduculate Oak Acorns (Quercus robur L.) from Spačva Basin and the Consequences of its Inadequate Storage

Kompleks šuma hrasta lužnjaka Spačvanskog bazena smjestio se u najistočnijem dijelu Hrvatske, u području rijeke Bosut i njezinih pritoka te čini petinu svih lužnjakovih šuma u Hrvatskoj. Hrast lužnjak gospodarski je vrlo značajna vrsta drveća. U uvjetima narušene ekološke ravnoteže vitalitet lužnjakovih sastojina značajno je narušen. Rezultati djelovanja negativnih činitelja vidljivi su kroz neredovit i sve slabiji urod sjemena i narušenu prirodnu obnovu šuma, a zbog pomanjkanja odgovarajućeg reprodukcijskog materijala otežano je obavljanje radova biološke reprodukcije. Stoga je potrebno u godini dobrog uroda sakupiti sjemenski materijal te ga uskladištiti na način koji omogućava da se zadrži njegova visoka kvaliteta. Uvjeti skladištenja žira na terenu su raznoliki i često neodgovarajući što ima za posljedicu značajno smanjenje kvalitete žira, odnosno njegovo propadanje. Prema načinu skladištenja žir spada u grupu rekalcitrantnog sjemena. Ciljevi ovog istraživanja su utvrditi kvalitetu svježe sabranog žira i žira nakon neodgovarajućeg skladištenja te zdravstveno stanje i vrste patogenih gljiva koje se razvijaju u i na žiru tijekom skladištenja. Uzorci žira sakupljeni su na pokusnim plohama u fiziološki zrelim sastojinama različite dobi. Kvaliteta sjemena je određena ISTA metodama, a zdravstveno stanje sjemena fitopatološkim metodama. Klijavost svježe sabranog žira bila je visoka, od 78 % do 97 %, a zdravstveno stanje okularno dobro sa slabo izraženim simptomima gljivičnih bolesti i oštećenjima od insekata. Nakon pokusnog skladištenja netretiranog žira u neodgovarajućim uvjetima u trajanju od godinu dana klijavost se znatno smanjila i iznosila je od 6 % do 16 %, a zdravstveno stanje sjemena je bilo izrazito slabo. Udio bolesnog i gnjilog žira bio je od 84 % do 94 %. Na bolesnom žiru utvrđene su patogene gljive iz rodova: Penicillium, Phomopsis, Ophiostoma, Ciboria, Fusarium, Alternaria i Stereum. Ovo istraživanje će poslužiti kao baza za definiranje smjernica za zaštitu žira i mogućnost dužeg skladištenja.The largest forest complex of pedunculate oak Spačva is situated in eastern part of Croatia in the river basin Spačva and Studva. It occupies area of 39 789 ha, and makes one fi fth of all pedunculate oak forests in Croatia. Pedunculate oak is a climatogenic species and it constitutes principal forest communities. Results of all the negative pressures are visible through irregular and weaker crop of seeds and disturbed natural forest regeneration. Natural regeneration is hampered due to the lack of adequate reproductive material. Collecting seed material in good crop year and ensuring high quality storage of acorns provides the required amount of seeds for successful natural regeneration. The objectives of this study were to determine the quality of freshly harvested and inadequate storaged acorns, and the health condition and types of pathogenic fungi which infected acorns during storage. Samples were collected on experimental plots in physiologically mature stands of diff erent ages. Acorn quality was determined by the ISTA method and the health status of seed by the phytopathological research methods. Germination of fresh acorns was high, varies from 78 % to 97 %. Health condition of acorns was good with weak symptoms of fungal diseases and insects damages. After storage of untreated acorns under unfavorable conditions for a period of one year germination decreased significantly and ranged from 6 % to 16 %. The share of the disease and rotten acorn was 84 % to 94 %. Seven genera of fungi were detected on inadequate storaged acorns: Penicillium, Phomopsis, Ophiostoma, Ciboria, Fusarium, Alternaria and Stereum

Eutypella parasitica R.W. Davidson & R.C. Lorenz SEVEN YEARS AFTER THE FIRST FINDINGS IN CROATIA

Gljiva Eutypella parasitica uzročnik je bolesti raka kore javora. Ta bolest umanjuje ekonomsku i estetsku vrijednost javora. Na zaraženim dijelovima stabala dolazi do truljenja drva, što oslabljuje statiku stabla i predstavlja opasnost od loma.U Europi je bolest raka kore javora prvi puta registrirana u Sloveniji 2005. godine. Od 2005. do 2008. godine bila je na EPPO Alert listi zbog opasnosti od širenja zaraze.U Hrvatskoj je bolest rak kore javora registrirana prvi puta 2007. godine na A. campestre u blizini granice sa Slovenijom. Od 2011. godine u Hrvatskoj se provodi program Poseban nadzor gljive E. parasitica.Cilj ovoga rada je utvrditi stanje zaraze rakom kore javora sedam godina nakon prvog nalaza, rasprostranjenost bolesti i intenzitet zaraze u Hrvatskoj. Drugi cilj je potvrditi vrstu gljive fitopatološkom i molekularnom analizom te pohraniti njihove DNA sekvence ITS2 regije u GenBank (NCBI).U razdoblju od 2011. do 2014. godine pregledane su šumske sastojine u kojima su zastupljena stabla javora na području 8 županija i 17 lokaliteta. Od ukupno pregledanih 2209 stabala, svega 2 stabla (A. pseudoplatanus i A. campestre) u blizini mjesta Hum na Sutli uz slovensku granicu bila su zaražena rakom kore, što iznosi 0,09 % zaraženih stabala. Zaraza rakom kore javora je vrlo malog intenziteta i bolest se nije proširila na druge lokalitete. Na uzorcima rakastih tvorevina laboratorijskim analizama, fitopatološkom analizom i DNA analizom dijagnostičkim početnicama, potvrđena je prisutnost gljive E. parasitica. Fragmneti DNA su sekvencirani te je napravljena usporedba u genskoj bazi podataka (GenBank, NCBI) gdje je potvrđeno da se radi o 100 % istovjetnosti s E. parasitica. ITS2 regija gljive E. parasitica utvrđena na gorskom javoru i na klenu, sekvencirana je i pohranjena u gensku banku (GenBank, NCBI) pod pristupnim brojevima KP868619 i KP868620.E. parasitica je prvi puta utvrđena u Hrvatskoj na klenu (A. campestre) 2007. Godine 2013. prvi puta je utvrđena u Hrvatskoj na gorskom javoru (A. pseudoplatanus), a 2014. godine potvrđena je na klenu (A. campestre). Vrsta gljive E. parasitica potvrđena je fitopatološkom i molekularnom DNA detekcijom. Zaraza rakom kore javora nije se proširila zadnjih sedam godina u Hrvatskoj.Fungus Eutypella parasitica is cause of bark canker on on maple trees. The disease reduces the economic and aesthetic value of maple. In Europe, the Eutypella canker was first recorded in Slovenia in 2005. From 2005 to 2008 the disease was listed on the EPPO Alert List because of the danger of contagion. In Croatia, Eutypella canker was recorded for the first time in 2007 on A. campestre near the border with Slovenia. The program Special monitoring of fungus E. parasitica was carried out since 2011 in Croatia. The aim of this study was to determine the status of infection on maples seven years after it was recorded for the first time, spread of the disease and infection intensity in Croatia. The second objective was to confirm the type of fungus using phytopathological and molecular analysis and store the DNA sequence of ITS2 region in GenBank (NCBI). The forest stands that represent maple trees in the area of 8 counties and 17 localities were examined in the period from 2011 to 2014. From a total of 2209 examined trees, only 2 trees of A. pseudoplatanus,and A. campestre, in the vicinity of settlement Hum na Sutli on the Slovenian border, were infected by the fungus, representing only 0.09 % of infected trees. Eutypella canker was found with very low intensity and the disease has not spread to other sites. The samples of cankerous formations were submitted to laboratory analysis, morphological analysis, and DNA analysis to confirm the presence of fungi E. parasitica. Fragments of DNA were sequenced and compared with gene in databases GenBank (NCBI), where it was confirmed that it was a 100 % identity with the E. parasitica. ITS2 region fungus E.parasitica established as sycamore and chubs was sequenced and deposited in gene bank (GenBank, NCBI) under accession numbers KP868619 and KP868620. Fungus E. parasitica was recorded for the first time in Croatia on A. pseudoplatanus in 2013, and its presence was confirmed on A. campestre in 2014. Type fungus E. parasitica was confirmed by morphological and molecular DNA detection. Bark canker on maples has not been significantly expanded during the last seven years in Croatia

Dynamics ofAcorn Production and Quality of English OakAcorn (Quercus robur L.) in Disrupted Ecological Conditions

Hrast lužnjak je klimatogena vrsta i tvori trajne šumske zajednice. Teško podnosi promjene u stanišnim uvjetima koje se događaju zbog nepovoljnih biotskih i abiotskih čimbenika. U današnjim uvjetima narušene ekološke ravnoteže vitalitet lužnjakovih sastojina je narušen. Znaci propadanja postaju vidljivi, a očituju se kroz narušen vitalitet stabala, neredovit i sve slabiji urod sjemena te narušenu prirodnu obnovu šuma. Varijabilnost plodonošenja žira rezultat je genetičke konstitucije vrste i sinekoloških faktora. Plodonošenje hrastova je pod genetičkom kontrolom preko majčinskih stabala. Hrast lužnjak, vrsta iz podroda Lepidobalanus ima tendenciju periodičnog uroda. Urod se pojavljuje u intervalima svake druge godine, a obilan urod jedanput u 4 godine. Istraživanja varijabilnosti plodonošenja lužnjaka provedena su na području Spačvanskog bazena u razdoblju od 2006. do 2010. godine. Cilj istraživanja bio je praćenje uroda po količini i kvaliteti u sastojinama različite dobisrednjedobnim, starijim i starim sastojinama, kako bi se utvrdio proizvodni potencijal sastojina. Pokusne plohe osnovane su 2006. godine. Količinska procjena uroda – rasta i razvoja žira, praćena je metodom sjemenomjera koji su postavljeni ispod stabala različitog stupnja osutosti krošanja. U godinama uroda kontrolirano je sakupljan žir, a u proljeće sljedeće godine izbrojen ponik. Sakupljeni biljni i sjemenski materijal obrađivan je u Laboratoriju za ispitivanje kvalitete sjemena. Kvaliteta sjemena ispitana je prema ISTA metodologiji. Dinamikom praćenja rasta i razvoja zametnutog žira metodom sjemenomjera, ustanovljeno je da je najviše normalno razvijenog i dozrelog žira bilo u starijim sastojinama, a najmanje u srednjedobnim sastojinama. Glede stupnja osutosti krošanja, najviše dozrelog žira nalazilo se u krošnjama stabala stupnja osutosti 2A, a najmanje u stupnjevima osutosti 2B i 3. Prvi urod žira pojavio se 2006. godine, a drugi 2010. godine. U 2006. godini urod je bio obilniji. Najbolje su urodile stare sastojine (prosječno 269 kg/ha), a najslabije srednjedobne (41kg/ha). Sa smanjenjem starosti, količina sakupljenog žira se smanjivala. U 2010. godini najviše žira sakupljeno je u starijim sastojinama (prosječno 90 kg/ha), u srednjedobnim sastojinama sakupljena je najmanja količina (30 kg/ha). Prosječan broj ponika bio je najveći u starim sastojinama (40.000kom/ha), a najmanji u starijim (16167 kom/ha). Periodicitet plodonošenja u razdoblju od 11 godina, na području g.j. Slavir i na području cijeleUŠP Vinkovci, imao je isti trend. U jedanaestogodišnjem razdoblju praćenja plodonošenja, rodne godine bile su: 2000, 2002, 2003, 2006. i 2010. godina. Kvaliteta sjemena u godinama uroda bila je dobra. Prosječne vrijednosti kvalitete sjemena sakupljenog na plohama bile su: urod 2006. godine – vitalitet 88 %, prisutnost insekata 12 %, masa 1000 sjemenaka 5384 g, broj žira/kg 185 kom/kg; urod 2010. godine – klijavost 71 %, bolesno i gnjilo sjeme 29 %, masa 1000 sjemenaka 4933 g, broj žira/kg 203 kom /kg. Na temelju provedenog istraživanja dinamike plodonošenja i kvalitete uroda sjemena, može se zaključiti da vitalitet krošanja značajno utječe na količinu proizvedenog sjemena, da su stare sastojine proizvele najveću količinu žira, a da se proizvedena količina žira smanjivala sa starošću sastojina. Broj ponika, nakon godine dobrog uroda, ukazuje na činjenicu da u krošnjama ostaje najkvalitetniji žir, koji polako otpada sa stabala i ostaje u sastojini nakon što je komercijalno sakupljanje žira završeno. U srednjedobnim sastojinama broj ponika je dvostruko veći nego u ostalim istraživanim sastojinama, vrijeme dozrijevanja žira u krošnjama je najduže, a žir otpada kasnije. Praćenjem dinamike i količine plodonošenja u razdoblju od 11 godina, urod žira se pojavljivao u različitim količinama. U tom intervalu bilo je 5 rodnih godina. Obilnost uroda predstavlja proizvodni potencijal sastojine. U starim sastojinama u godinama dobrog uroda ustanovljen je prosječan urod od 269 kg/ha, odnosno 114742 komada žira/ha. Obilnost uroda je daleko od onog što se smatra obilnim urodom. Stoga će biti potrebno uložiti veliki napor da se urod očuva i u krošnjama i nakon dozrijevanja i opadanja.Pedunculate oak is a climatogenic species and it constitutes principal forest communities. If some ecological factor in its habitat is altered, due to unfavorable biotic and abiotic factors, common oak cannot adjust fast and it suffer substantial damage over the years. Current ecological imbalance can be recognized as main reason in oak dieback. Deterioration sings become visible and they are reflected in decline of tree vitality, unsettled and low yield and also remote natural forest regeneration. Variability in acorn production is the result of sin ecological and genetic factors. Acorn yield is the most influenced by maternal trees. Pedunculate oak belongs to subgenus Lepidobalanus and has tendency of periodical yield, with return of every 4 years (abundant), and every 2 years (normal). Research on acorn production have been carried out in “Spačvanski bazen” area in period from 2006 to 2010. Main research goal was to monitor acorn crop according to quality and quantity, in stands of different age - middle aged, older and old forests, so production potential could be identified. Experimental plots were established in 2006. (Tables 1 and 2). Acorn production assessment- acorn growth and development, was monitored by cone-shaped acorn-collecting traps method. Acorn-collecting traps were placed beneath the canopy of selected trees of different crown damage classes. In the crop years acorns were collected, and in the spring next year seedlings were counted. Collected materials were analyzed in the Laboratory for testing seed quality. Seed quality was tested accordingly to ISTA methodology. Monitoring of growth and development of acorns by acorn-collecting traps method (Table 3) proved the fact that most matured and normally developed acorns came from older stands, and the least in old stands. Regarding crown damage degrees the most matured acorns came from crown damage class 2A, and least from class 2B and 3. First acorn crop was in 2006 and second in 2010. (Tables 4 and 5). In year 2006 acorn crops was more abundant than in 2010. The best acorn crop was in old stands (on average 269 kg/ha), the worst was in middle aged forests (41 kg/ha). With reduction of tree age came reduction in acorn crop. Yield in 2010 was the best in older forests (90 kg/ha) and the worst in middle aged forests (30 kg/ha). Average number of seedlings was at its peak in old forests (40000 pts/ha), the worst in older forests (16167 pts/ha). Periodicity in acorn production during the period of 11 years in the management unit Slavir and on the area of Forest Administration Office Vinkovci followed same trend in 11 years time (Figure 1 and 2). Annual acorn production was monitored for 11 years and crop years were: 2000, 2002, 2003, 2006 and 2010. Average quality values for acorns harvested on experimental plots were: 2006. (Figure 3)- viability 83%, insects presence 12% 1000 seed weight 5384 g, number of acorn/kg 185 pts/kg; yield 2010. (Figure 4): germination capacity 71%, rotten and decayed seed 29%, 1000 seed weight 4933 g, number of acorn/kg 203 pts/kg. Based on study of acorn periodicity yielding and quality crops conclusions can be made: degree of crown defoliation significantly influences quantity of produced acorns, old forests produced the most acorns, seed production decreased with age. Number of seedlings shows us that the most quality acorns remains in crowns and its fells after commercial seed collecting are completed. In middle aged forest number of seedlings in double the quantity than other investigated stands, acorn maturation is longest, and acorn fells on ground later. Monitoring of dynamics and seed production quantity during 11 years, crop years occurs significantly every 2 to 3 years and they differ in quantity. During that period were 5 mast years. Abundance of yield represents productional potential of stands. In old stand abundant acorn crops is identified with average yield of 269 kg/ha or 114748 pts of acorn /ha. Seed production abundancy is far below what is considered as abundant, therefore significant effort is required for preserving acorns in crowns after maturing and felling on the ground

Review of investigation of variability of nad1 gene intron B/C of mitochondrial genome in Scots pine (Pinus sylvestris L.)

Background and Purpose: Scots pine (Pinus sylvestris L.) is the most extensively distributed pine, covering the whole Eurasian continent. The detection of genetic variability of Scots pine is of greate importance from the evolutionary point of view as well as for genetic conservation. Mitochondrial genome in Scots pine is maternally inherited and dispersed through seeds. Molecular studies have indicated high genetic diversity in Scots pine genome, allowing the detection of variation between and within population. Mitochondrial genome possesses variable regions in the gene encoded NADH dehydrogenase. The variable region is nad1 B/C intron. The different haplotypes of nad1 B/C intron allowed confirming the Iberian refugia in Holoecen and confirming the genetic diversity between different stands of Scots pine in Poland. The purpose of this study was to find out the genetic variability of nad1 B/C intron in two International Scots pine provenance experiments and native Scots pine provenance in Croatia in order to detect multiple origin and haplotype variation between provenances. Material and Methods: The samples of Scots pine were taken from two International experiments of provenances, one established in Croatia (22 provenances) and the other established in Hungary (20 provenances) and Croatian native provenance (Forest office Vrhovine, Forest administration Gospić). The samples of needles were collected from randomly chosen trees of each provenance. Eight samples per provenance were collected for haplotype analyses. DNA was extracted and amplified with specific primers for nad1 B/C intron of the mitochondrial DNA. PCR products were analyzed using agarose gel electrophoresis and capillary electrophoresis on the bioanalyzer Agilent 2100. Results and Conclusion: We investigated samples from 42 provenances of Scots pine that originated from a broad range in Eurasia and were included in two international provenance experiments, and samples from native Croatian population. A total of 344 individual trees of Scots pine were analyzed. The datawere analyzed by agarose gel electrophoresis and by capillary electrophoresis. We did not detect variability in mitochondrial nad1 B/C region in analyzed samples. All analyzed samples were haplotype a. Based on our data, we could conclude that none of the tested samples originated from the Iberian glacial refugia. All tested samples could have the origin in glacial refugia placed in Mediterranean, Balkan region or in the northeastern Europe. More variable region of the nuclear DNA and themitochondrial DNA should be tested on a greater number of samples to obtain more informative data