Zbornik gozdarstva in lesarstva, 54, 1997, s. 109 - 124 GDK 411.16: 172.8 (Cenangium ferruginosum Fr.) +181.22 +181.31 Prispelo/ Received: 10.10.1997 Sprejeto/ Accepted: 28. 1 O. 1997 Izvirni znanstveni članek Original scientific paper INFLUENCE OF MOISTURE, TEMPERATURE AND GROWTH MEDIUM ON CENANGIUM DIEBACK OF PINE (CENANGIUM FERRUGINOSUM FR., ASCOMYCOT/NA) Dušan JURC., Maja JURc·· Abstract C. ferruginosum culture was grown in vitro on MEA medium at 1 O, 20, 25; 30 and 35° C, on media with water activity modified to 0,98, 0,96 and 0,94 and on MEA, PDA and OA media at 23° C. The fungus grows at temperatures between 1 o° C and 25° C and on media with available water between 0,98 and 0,96. The optimal temperature for growth is 25° C, while at 30° C it doesn't grow at all. The best growth occurs on PDA medium, the second best on MEA medium and the lowest growth on OA medium. Composition of growth medium is an important factor and influences growth rate considerably. These results explain the ability of the fungus to invade the tissues of its host during colder periods of year and when the substrate is highly moistened. High summer temperatures are unsuitable for its growth. Keywords: Cenangium dieback of pines, Cenangium ferruginosum Fr., temperature, moisture, growth medium. VPLIV VLAGE, TOPLOTE IN HRANILNE PODLAGE NA SUŠICO BOROVIH VEJ (CENANGIUM FERRUGINOSUM FR., ASCOMYCOTINA) Izvleček lzolat C. ferruginosum smo gojili na hranilni podlagi MEA pri 1 O, 20, 25, 30 in 35° C, na gojiščih z vodno aktivnostjo uravnano na 0,98, 0,96 in 0,94 in na hranilnih podlagah MEA, PDA in OA pri 23° C. Gliva uspeva na temperaturah od 10° C do 25° C in na hranilnih podlagah z vodno aktivnostjo med 0,98 in 0,96. Optimalna temperatura za rast je 25° C, vendar pri 30° C ne uspeva več. Najbolje raste na hranilni podlagi PDA, manj na MEA in najmanj na OA. Sestava hranilne podlage je pomemben dejavnik, ki močno vpliva na hitrost rasti glive. Ugotovitve pojasnjujejo sposobnost glive, da prerašča tkiva gostitelja v hladnejših letnih obdobjih in ob visoki vlažnosti substrata. Visoke poletne temperature so neustrezne za njeno rast. Ključne besede: sušica borovih vej, Cenangium ferruginosum Fr., toplota, vlaga, hranilna podlaga. Asist. mag., dipl. biol., Gozdarski inštitut Slovenije, Večna pot 2, 1000 Ljubljana, SLO •• Asist. dr., dipl. inž. gozd., Gozdarski inštitut Slovenije, Večna pot 2, 1000 Ljubljana, SLO 110 Zbornik gozdarstva in lesarstva, 54 CONTENTS / KAZALO 1 INTRODUCTION / UVOD ................................................................. 111 2 MATERIAL AND METHODS / MATERIAL IN METODE ............ 112 2.1 COLLECTING SITE/ MESTO VZORČENJA ................................ 112 2.2 SAMPLING PROCEDURE ANO ISOLATION / NAČINI VZORČENJA IN IZOLACIJA ............................................................ 112 2.3 MEASURMENT OF FUNGAL GROWTH IN VITRO I MERJENJE RASTI GLIVE IN VITRO ............................................. 113 3 RESULTS / REZULTATI .................................................................. 113 3.1 EFFECTS OF TEMPERATURE ANO AVAILABLE WATER ON GROWTH OF C. FERRUGINOSUM I VPLIV TEMPERA TURE IN DOSTOPNE VODE NA RAST C. FERRUGINOSUM ............................................................................. 115 3.2 DIFFERENCE IN GROWTH ON MEA, PDA ANO OA / RAZLIKA V RASTI NA MEA, PDA ANO OA .................................. 118 4 DISCUSSION / RAZPRAVA ............................................................ 119 5 POVZETEK ......................................................................................... 121 6 ACKNOWLEDGEMENTS / ZAHVALA .......................................... 122 7 REFERENCE$/ VIRl ........................................................................ 122 1 INTRODUCTION 111 Jurc D., Jurc M.: Vpliv vlage, toplote in hranilne podlage ... Austrian pine grows in very small scattered natura! stands in the Alpine regions and also in central and southern parts of Slovenia. It is widely distributed as an allochtone tree species in the Karst region near the Adriatic coast, where it has been planted on more than 20.000 ha of degraded land beginning more than 100 years ago. The epidemic of Cenangium ferruginosum Fr. in 1986 affected the greater part of the region and caused the felling of approximately 10.000 m3 of Austrian pine (JURC 1987). Although only trees with more than 80 % of the crown dead were cut, numerous trees with dead branches in the crowns remained. Since that tirne the Cenangium dieback of pines has only appeared occasionally and locally. Analysis of climatic factors for 1985, in comparison with 1 O year averages, showed that the relative humidity had reached a 1 O year minimum in all months from July to October, registered precipitations reached a 1 O year minimum in September and October, while in August temperatures reached a 1 O year maximum. This unusually dry, hot weather was proceeded by an unusually wet period. The first signs of dying in Austrian pine branches were noticed in December 1985. The largest mortality occured in 1986 on the southern and south-western slopes and in areas with shallow soil. AII observations were in accordance with other well-described examples from the literature where drought stress is regarded asa decisive factor in C. ferruginosum dieback (JURC 1987). C. ferruginosum is usually regarded as ubiquitus and opportunistic fungus which kills the bark and cambium of twigs and branches weakened by environment, other pests and pathogens, or natural senescence (BUTIN 1996, SINCLAIR / LYON/ JOHNSON 1987). Outbreaks of Cenangium dieback occur only once in several years. The fungus is usually a competent saprobe on pine twigs killed by various agents. lts saprobic existence ensures a supply of spores and it seems likely that incipient infections begin each year in the summer and autumn and are held in check by host defenses unless these are overcome by environmental damage or by other pests (SINCLAIR/ LYON / JOHNSON 1987). This common view of repeated yearly infections was overturned by the results of JURC (1996) who revealed the constant endophytic existence of C. ferruginosum in the healthy needles of Austrian pine. 112 Zbornik gozdarstva in lesarstva, 54 The objectives of our laboratory study were to determine the temperature and available water requirements for the growth of C. ferruginosum in culture and to test its growth on different artificial nutrient media. We hoped that these data would reveal the ecological factors which are decisive in fungal growth and are prerequisites for outbreaks of the disease. Such factors should enable the fungus to break its quiescent endophytic state and to spread quickly through weakened tissues of its host. 2 MATERIAL ANO METHODS 2.1 COLLECTING SITE A sampling of Austrian pine needles was performed in a natura! stanci of Austrian pine on Smolnik hill near the town of Jesenice (1075 m above sea level, y=54 343000, x=51 42100; projection G.-K., UTM MV43) in January 1995. This forest belongs to plant association Pinetum subil/yricum Schmidt 1936 and grows on limestone. 2.2 SAMPLING PROCEDURE ANO ISOLATIONS The tree from which the samples were taken was about 60 years old. A branch was cut from the lower whorl of the southern part of the crown using tree loppers. Needles were transported in cold boxes at 4°C in clean polyethylene bags and processed within 24 hours. Four needles of each age class, without any signs of damage, were washed in running tap water for one hour. From every needle three 0,3 cm long segments were taken - one from the base, one from the middle part and one from the tip of the needle. They were then surface-sterilised (1 min in 50 % ethanol v/v, 5 min in sodium hypochlorite with 2,6 % of active chlorine, 1 min in 50 % ethanol v/v). The segments were blotted dry and plated onto 2 % malt extract agar (Malt Extract, Biolife S.r.l., 20 g r1, Agar Bios Special LL, Biolife S.r.l., 20 g r\ Twelve segments per plate and 2 replicate plates per age class were plated out. Petri dishes were then incubated at 23° C and examined weekly for six weeks. Mycelial outgrowths from the segments were subcultured and identified. 113 Jurc D., Jurc M.: Vpliv vlage, toplote in hranilne podlage ... The fungus used in all further experiments was isolated from the base segment of a four year old needle and it fit, in all details, to the description of C. ferruginosum anamorf (KUJALA 1950). 2.3 MEASURMENT OF FUNGAL GROWTH IN VITRO 1 % malt extract agar medium was used (MEA, Lab M Ltd, pH 4,5) to measure fungal growth. In all cases 25 ml of malten agar was poured into 9 cm diameter Petri dishes and inoculated with 4 mm diameter agar plugs of C. ferruginosum cultures taken from the growing margin of 14 day old colonies. The diameter of the colonies was measured in two directions at right angles to each other. AII experiments were carried out in triplicates. The effect of temperature was examined by placing inoculated MEA agar plates in darkness at 1 O, 20, 25, 30 and 35° C. The effect of water availability (aw) was examined by modifying the media with glycerol to the values of 0,98, 0,96 and 0,94 aw (DALL YN / FOX 1980, MAGAN / LACEY 1984). Plates with the same aw were enclosed in clean polyethylene bags and incubated at 1 O, 20, 25, 30 and 35° C for 29 days. To compare the growth rate on different growth media the fungus was incubated at 23° C on 2 % MEA (Malt Extract, Biolife S.r.l., 20 g r1, Agar Bios Special LL, Biolife S.r.l., 20 g r\ 3,9 % PDA (Potato dextrose agar, bioMerieux 51411, 39 g r1) and 3 % OA (oatmeal 30 g r1, Agar Bios Special LL, Biolife S.r.l., 20 g r1) 3 RESULTS Colonies of C. ferruginosum in culture have characteristics which make the growth tests less comparable and reliable. The growth of the mycelial colony is irregular and outgrowths can develop from some parts while in other parts the growth can be slower or halted. This is the reason for the great variation seen among some of the experiments. Photo 1 shows the morphology of C. ferruginosum cultures on MEA, OA and PDA media, while photo 2 shows slimy conidial masses on mycelium. 114 Zbornik gozdarstva in lesarstva, 54 Photo 1: Differences in growth of 41 days old C. ferruginosum cultures on OA, MEA and PDA media (from left to right). Fotografija 1: Razlike v rasti 41 dni starih kultur C. ferruginosum na gojišču OA, MEA in PDA (od leve proti desni). Photo 2: Slimy conidial masses on culture of C. ferruginosum (ali photos by D. Jurc). Fotografija 2: Sluzaste mase konidijev na kulturi C. ferruginosum (vse fotografije: D. Jurc). 115 Jurc D., Jurc M.: Vpliv vlage, toplote in hranilne podlage ... 3.1 EFFECTS OF TEMPERATURE ANO AVAILABLE WATER ON GROWTH OF C. FERRUGINOSUM At 1 o° C and 20° C the growth of C. ferruginosum is similar, while the highest growth rate occurs at 25° C. High temperatures (30° C and 35° C) are not suitable far the growth of the fungus, in fact, at these temperatures, it doesn't grow at all (Graph 1). 62 58 E 54 §_ 50 ~ .a 46 3 -"' cii 42 E 38 ~ a.. - 34 E 30 §_ ~ 26 .a 3 22 (.) o 18 * 14 E lt1 10 o 6 2 Graph 1: Grafikon 1: --------------------------------------··1~ r -ff;:f ::-.::-.::-. - - - - .-:-:::::::.-::-:::.-::-.::-.::-.7 .-r··-_ _., z--,, ,,,~..::z.;. ....... CF _10 --- CF _20 ------ CF _25 ....... CF _30 3 5 7 9 11 13 15 17 19 21 23 25 27 29 --- CF - 35 Days of growth / Dnevi rasti Diameter (mean ± S.E.) of C. ferruginosum culture at temperatures 1 O, 20, 25, 30 and 35° C. Premer (srednja vrednost ± S.E.) kulture C. ferruginosum pri temperaturah 1 O, 20, 25, 30 in 35° C. 116 Zbornik gozdarstva in lesarstva, 54 At 10° C C. ferruginosum grows well on relatively humid growth medium (aw1, aw2), it doesn't grow in relatively dry conditions (aw3) (Graph 2). 60 E _§__ ~ 50 .a =i -"' cii 40 E ~ C.. -E 30 _§__ ~ .a =i 20 o o ~ * E 1 O "' i5 _yr· , --/ 1------_;c -1 T-'- / .. --"'"' -----------------! ----------1 ---- ---:.L---_.:;c ~----------------------------------------------------- ··•··•· AW1_10 o Graph 2: Grafikon 2: --- AW2_10 3 5 7 9 11 13 15 17 19 21 23 25 27 29 -··-·· AW3_10 Days of growth / Dnevi rasti Diameter (mean ± S.E.) of C. ferruginosum culture on medium with different available water (aw1 = 0,98, aw2 = 0,96, aw3 = o 0,94), at 10 C. Premer (srednja vrednost ± S.E.) kulture C. ferruginosum na podlagi z različnimi količinami dostopne vlage (aw1 = 0,98, aw2 = 0,96, aw3 = 0,94), pri 10° C. 117 Jurc D., Jurc M.: Vpliv vlage, toplote in hranilne podlage ... Growth rate of the fungus at 25° C and higher aw (aw1, aw2) is less than that at 1 o° C. As at 1 o° C the fungus doesn't grow at aw3 (Graph 3). E .s w ::i '§ -" ai E e! a.. -E .s e! .2 :5 u o 2 w E