ALEPPO PINE DEFENCE AGAINST SPHAEROPSIS SAPINEA, CRONARTIUM FLACCIDUM AND OTHER FUNGAL PATHO- GENS IN THE MEDITERRANEAN PART OF MONTENEGRO

s: Aleppo pine (Pinus halepensis Mill.) is the most widely disturbed conifer tree in native forest and plantations throughout the Adriatic coast of Montenegro. However, pathogenic fungi in the region are extremely diversified and constitute the principal threat to healthy tree growth: 15 pathogenic fungal species have beden identified in the Eumediterranean zone a typical habitat of Aleppo pine, whereas 21 fungal pathogens occurred in northern Submediterranean zone an atypical habitat for Aleppo pine in Montenegro. Greatest damages are inflicted by Sphaeropsis sapinea, a pathogenic generalist on pine, and Cronartium flaccidum, causal agent of rust disease on pine. The following relates, for the first time, the presence of hypersensitive response (HR) in young needles and shoots (organs rich in nitrogen) as a specific defense mechanism in Aleppo pine against Sphaeropsis sapinea, an otherwise very harmful pathogen for Austrian pine (Pinus nigra Arn.) and other pines in the Mediterranean part of Montenegro. Thus, better understanding of the ecology of S. sapinea, of C. flaccidum, as well as of associated mycobiota and mechanisms of host defense are essential for establishing measures and tools for protection of Aleppo pine ecosystems.


INTRODUCTION
Aleppo pine (Pinus halepensis Mill.) is a native coniferous tree of the Mediterranean region.Its extends to Morocco and northern Spain, southern France, Italy, eastern Adriatic coast, Greece and northern Libya (Fa r j o n , 2005).It is generally found at low altitudes, mostly from sea level in the Eumediterranean zone (ass: Oleo lentiscetum and Pinetum halepensis) to 300 m altitude in the Submediterranean zone (ass: Rusco-Carpinetum orientalis and Ostryo-Quercetum pubescentis) of Montenegro (Vu j a n o v i ć , 1990).According to J o v a n o v i ć (1971), Aleppo pine is a small to medium-size tree, reaching 18 m of height.It is an important tree species in the Mediterranean region, being a major component in forestry, in ornamentation and landscaping as well as in parks and gardens in hot/dry areas-due to its considerable heat and drought tolerance.Furthermore, as a result of its tolerance to aforementioned abiotic stresses, Aleppo pine demonstrates relatively moderate forest die-back damage in Montenegro, with only a 16% decline (Vu j a n o v i ć , 1990).However, there are several other abiotic as well as biotic stressors affecting Aleppo pine ecosystems.Each year large areas are destroyed by wildfire and soil erosion.Frequent outbreaks of pathogenic fungi and insects also present a threat.Nevertheless, despite the importance to study Aleppo pine diseases in the Mediterranean part of Montenegro, no extensive research has been done in this regard.
Hence, this paper aims to provide more light on major fungal diseases and to elucidate their potential significance in the overall process of tree dying.More specifically, the present manuscript wishes to relate and improve our understanding of a defense mechanism unique to Aleppo pine as opposed to others members of the pine genus: the hypersensitive response.As such, Aleppo pine is noted to be less diseased or symptomatic than Austrian pine (Pinus nigra Arn) althought these species grow in proximity or even together (mixed plantations).All this information are pertinent for better, future disease prevention.

Fungal diversity study
The research was carried out in Aleppo pine native forests and plantations in both the Eumediterranean (Bar and Sutomore) and Submediterranean (Podgorica, Virpazar) zones of Montenegro.Plant samples were collected in different period of the year, between 1991 and 1992; fungal taxa diversity from samples was then analyses in laboratory.
Using Carl Zeiss microscope, fungi were identified based on phenotypic characters (fruiting bodies, spores, spore bearing and reproduction organs) as well as fungal pure cultures.Almost all observed fungi (except rusts) were isolated on nutritive maltextract (MEA, Becton Dickinson) and potato-dextrose (PDA, Becton Dickinson) agar media (B o o t h , 1971).All isolates were examined for rate of colony growth and for the presence of fructifications produced in culture.
The identification was performed using the following taxonomical keys: B u t i n (1989), D e n n i s (1978), E l l i s and E l l i s (1985), Ev a n s (1984), G r o v e (1935,1937), H a n l i n (1992,1998) (1978), M i n t e r at al. (1978), M i n t e r (1981) and Sutton (1980).

Sphaeropsis sapinea infection assays
In order to test the difference between Aleppo and Austrian pine tolerance to major disease agents, Sphaeropsis sapinea colonization efficiency was assessed in two, main needle/shoot phenophases or growth stages.
Series of comparative artificial inoculations on Aleppo and Austrian pine species were carried out using spore suspension in sterile distillate water (10-30 conidia per mL) followed by artificial infections of young needles/shoots in I-growth stages (needles reaching half of their normal length) and in older needles/shoots or II-growth stages (needles longer than half of their full, possible length); nitrogen (N) content in needles and shoots was simultaneously analyzed by Kheldal methods (1990).

RESULTS AND DISCUSSION
Research results are presented in Table 1.Data clearly shows that 21 pathogenic fungal taxa were associated to plantations in Submediterranean zone compared to only 15 taxa in the Eumediterranean.
Thus, results also indicate that Aleppo pine plantations grown in Submediterranean zone, or outside the tree,s typical Eumediterranean habitat, are more susceptible to fungal attack or with a higher risk for disease.Considering the level of importance or pathogenic significance of each fungal taxa on the health of Aleppo pine, listed fungi can be classified in three main pathogenic groups.
Group I -Represented by Sphaeropsis sapinea and Cronartium flaccidum Fungus S. sapinea was largely distributed and has been indentified in all studied locations, throughout the Eumediterranean and Submediterranean zones.In addition to Aleppo pine, it has been observed on Maritime pine and Austrian pine.Fruiting bodies (pycnidia) are found on short needles of shoots infected during the previous year, and on bark and scales of second-year seed cones.Pycnidia of this fungus were observed also on the root of maritime pines (Pinus maritima and P. pinea), where it caused forked fissures and death of vital parts of the plant.Disease was especially harmful to thin, pine roots in plantations established on conglomerate with a shallow soil layer.Root infection was caused by infected fallen needles, which bore numerous fructifications.Seed infection affects pine regeneration, thus having negative effects on nursery practices and efficiency of reforestation.
Artificial Aleppo pine infection with S. sapinea spores (conidia) was conducted at the time of budding phenophase and new shoot formation.Aleppo pine showed numerous disease symptoms: infected needles on new shoots discolored (turning tan, reddish or brown) though without observed needle cast; lateral shoots curved at places and died; various deformation of bark appeared following an intense excretion of resin; and resin flowing in summer through bark fissures formed a sticky/adherent mass on the shoots.
Finally, typical symptoms of infection appeared: fruiting bodies or pycnidia of the fungus.Moreover, according to K a r a d ž i ć and S t o j a d i n o v i ć (1988), this fungus has two infection periods: the first one is in the second half of April (this period is more significant and infections are probably through bark of young shoots), and the second one is in the first half of June (infection probably goes through young needles which are at that period not yet at full length).It has been hypothesized that some pollutants, such as ammoniumsulfate, stimulate the development of this fungus (Roelofs et al., 1985(Roelofs et al., , cit. H o r n , 1985) ) since they increase nitrogen content in needles (D e K a m et al., 1991).Knowing the existence of a correlation between nitrogen content and fungus development, the nitrogen content of different tree parts was determined (Table 2).In turn, infection was done in organs with highest nitrogen levels in order to observed trees reactions when fungus is given an optimal condition.Hence, artificial infection was done on Aleppo pine needles and young (rapid growing) shoots at the I-growth stage, where the highest nitrogen (N>4%) content is detected.Interestingly, once infection was induced, a typical host-cells hypersensitive response (HR) was observed.The response is a form of programmed cell death (PCD) which provided total shoot's resistance.PCD started around the initial infection site followed by tissue necrosis and rapid death of the whole young shoot.PCD was associated with restricted pathogen growth, thus, preventing fungal mycelium progression to older shoots.In contrast, infected Austrian pine shoots at I-growth stage, which have considerably lower nitrogen content (N<2.5%),showed prolonged development, stopping growth, approximately, at the middle of normal growth length.Furthermore, fungal mycelium, as opposed to Aleppo pine, developed further by undisturbed spreading in older shoots during the following years.Eventually, the latter lead to death of entire branch in Austrian pine.Hence, programmed cell death in Aleppo pine seemed to be the driving defense mechanism explaining, to some extent, the lower damage inflected observed by S. sapinea to Aleppo pine.
Under II-growth stage treatments, both pine species often showed necrosis; necrosis occurred on individual, pair of current-year needles.When the mycelium spreads from young Aleppo pine shoot (II-stage; N<3.4%) to the one-year old (infrequent), forked, 1-3 cm long fissures (at places) were observed though infected branches remained alive.This demonstrated localized infection with no further fungal propagation or tree death.Meanwhile, in Austrian pine, old branches died and tree growth was compromised.Exception in Aleppo above -demonstrated vigor was observed during regular dye-back monitoring in "Cemovsko Polje"-Podgorica.Indeed, considerable tree death in young Aleppo pine plantations were observed; these were explained by additional stress factors combined to that of the fungal disease and included high, gaseous hydrogen fluoride (HF) pollution (from Aluminum plant in proximity of plantation).It is also important to note that tolerance levels of S. sapinea to fluore pollutants remain unknown and would be interesting to test.
As evident from our results, S. sapinea is a very dangerous pathogen not only for Austrian pine but also for other pine species such as Aleppo -particularly when combined with additional stressors such as pollution and drought.Therefore, adequate protection or sanitation management programs should take in consideration all of these aspects for efficient disease control.Highest control of fungus can be achieved by Benlate (Benomil) and copper oxychloride when applied twice within intervals of 7 days (M a r i n k o v i ć , K a r a d ž i ć , 1987).
The fungus C. flaccidum causes Aleppo pine bark rust.In places of infection, a canker wound forms and resin excretion occurs.Fungus progressively encircles stem or branches, bringing about slow death.Usually individual branches die first; later, progressive infection of stem brings about tree death.This fungus has been identified in the Aleppo pine plantation in the vicinity of the Scadarsko Lake, where it is of major significance.Aecia of this fungus on the branches were observed at the beginning of April and were stimulated by high humidity and temperatures as they stimulate fungal growth and reproduction.
Group II -Represented by Cenangium feruginosum, Lophodermium seditiosum and Thyriopsis halepensis C. feruginosum often causes branch dying and massive tree dying during fungal outbreaks.There are different opinions about of the parasitism of this species.Some authors claim that it is a parasite of weakness, i.e. fungus preferentially attacks physiologically weakened trees.Others state that the fungus is a dangerous pathogen, especially under intense drought conditions during which it causes massive tree dying.According to K a r a d ž i ć et al. (1990), in continental Serbia (Zlatibor), C. feruginosum outbreaks and deaths of Scots (Pinus sylvestris L.) and Austrian pine are initiated by preceding abiotic and biotic stresses.Among climatic factors, the most influent was prolonged drought in the summer period 1984-l986.Intense droughts resulted in physiological weakening of trees and their predisposition to C. ferruginosum attacks.Outbreaks correlated with presence of an associate bark beetle infestation.Tree dying was most expressed in southern, warm, shallow and skeletal soils on serpentinite.C. feruginosum outbreaks were also registered on various Mediterranean pine species: Pinus maritime Lam., P. pinaster Soland, P. pinea L. and P. halepensis Mill.In Montenegro, subjected to a different infection pattern.First C. feruginosum outbreaks on the Mediterranean pines were observed between 1986 and 1998 in Mediterranean region of Montenegro.It followed the massive attack of gall midge Thecodiplosis brachintera Schw.Registered from 1984 to 1986, which provoked chlorosis in pine crown and premature needle falls (Vu j a n o v i ć , data not shown).Symptoms of C. feruginosum firstly occur on weakened trees and lower branches.Also, when sexual fructifications massively appear, it was often too late to protect plantations from death.
T. halepnsis has been observed on green needles (most often one-year old ones), but also on dead needles.The fungus is especially prevalent in plantations in locality "Ratac" near Bar, and in the plantation near Skadarsko lake (Virpazar).On green needles collected in the second.middle of April, ripe fructification with asci and ascospores were identified.This is opposite G l a v a š's (1983) observations who noticed, in the region of Dalmatia, occurrence of only conidial stages in the spring while the perfect stage appeared in autumn.According to M i j u š k o v i ć (1986), pycnidia of this fungus were also found in the middle of April, whereas the perfect stage was already developed at the end of May.This author also reported that the most numerous asci and ascospores were observed at the beginning of September.There are contradictions within literature data in terms of the fungal implications and significance for Aleppo pine growth.Q u e l l e t t e (1966) underlined that further research should be expected in order to ascertain that the fungus is harmful to Aleppo pine.On the other hand, M i j u š k o v i ć (1986) believes that the fungus is very dangerous; the damage it caused in 1982 and 1983 in plantation near Podgorica stand as proofs of its high pathogenicity.For that matter, we have found that the fungus produce active fruiting bodies (thyrothecia) on both green and necrotic needles.Although numerous fructifications were frequently found on green needles (epiphytic), no modification in epidermal or mesophyll cells had been observed.This could be explained by the fact that the fungus might profit from stoma openings for nutrients uptake.
L. seditiosum has been observed at all the localities.Apothecia occur on primary needles of naturally occurring seedlings, or on two-year old needles of grown trees.Together with other fungi (i.e.Lophodermium pinastri, Cyclaneusma niveum) it causes needle blight.In continental regions, primary infection occurs from the middle of August to end of September (sometimes middle October).
Group III -Other, less significant fungi on Aleppo pine tend be saprophytes Thyronectria balsamea (Ascomycota) and Phellinus pini (Basidiomycota) form fructifications on standing trees in Sutomore and Podgorica.Coleosporium tussilaginis (needle rust) and Mycosphaerella pini, occur quite rarely and they have been observed only near Virpazar; these do not cause great damage.An interesting facts is that the fungus Elytoderma torresjuanii, most harmful and widespread fungi on Dalmatian, Mediterranean pines according to G l a v a š (1988), has not been detected during our investigations in Montenegro.According to the same author, this fungus also cause damage to Pinus halepensis and P. pinea and infection can be over 50% of the overall tree-crown assimilation area.
, K i f f e r and M o r e l e t (2000), K i r k et al. (2001), L a n i e r et al.

Table 2 .
Analysis of nitrogen content in the needles/shoots of Aleppo pine (P.halepensis) and Austrian pine (P.nigra) Табела