Fungi associated with a decline of Pinus nigra in urban greenery

© Slovak University of Agriculture in Nitra Faculty of Agrobiology and Food Resources, 2015 http://www.fapz.uniag.sk/ DOI: 10.15414/afz.2015.18.02.36–43


Introduction
In recent years, among urban trees Austrian pine also called European black pine (Pinus nigra Arnold) belongs to the particularly affected tree species.At the same time with regard to the expected global climate change, this change may cause the pathogens to adapt to the previously resistant species of woody plants (Kolářík et al., 2005).Austrian pine is a bold-textured and urbantolerant pine having a broad-pyramidal growth habit with ascending branches and showy spring candles and often developing a flat-topped crown and ornamental bark with age.This evergreen tree in pine family is used either as a single specimen or in group or mass plantings as visual screens or windbreaks (Sinclair et al., 1987).
The occurrence of causal pathogens causes withering of branches and tree thinning in the lower parts of the tree crown as a beginning of woody plants weakening by the low temperatures in winter period and by the drought from the spring to summer season (Grove, 1922; James, 1984; Manoharachary et al., 2003; Karadžić and Milijašević, 2008).
The aim of this study was to research distribution, disease symptoms, some important characteristics in pure culture and distinctive morphological features of the most important parasitic fungi in Austrian pine.The presence of these fungi in host tissue of symptomatic trees has been described using classical phytopathological approaches and microscopical identification based on morphological keys.

Material and methods
From spring to autumn 2013-2014 needles of Pinus nigra with blight symptoms were collected at several locations from plants growing in private gardens and public greenery of the town of Nitra (Nitra-Zobor, Nitra-Chrenová, Nitra-Sihoť, Nitra-Kynek).Altogether 20 trees were studied.The age of evaluated trees was between 35-40 years.Samples were taken from some sections of trees with damaged needles from every locality.Every sample was cultivated on 30 Petri dishes (PD) with 3% PDA (potato-dextrose agar -complete name of medium, when first use) medium.The samples of material have been deposed at the Institute of Forest Ecology of the Slovak Academy of Sciences, Branch for Woody Plants Biology in Nitra.Classical phytopathological approaches were used to isolate and obtain pure hyphal cultures.Fungi were isolated from the needles first immersed for one minute into 70% alcohol and then for 15 minutes into sodium hypochlorite (1% available chlorine).After that, the needles were washed twice in sterilized distilled water and cut to fragments of 2-5 mm which were placed on the nutritive medium, potatodextrose agar (PDA).Petri dishes with the medium and host fragments were incubated at 24±1 °C and 45% humidity in dark in a versatile environmental test chamber MLR-351H (Sanyo).Pure fungal cultures were obtained using multiple purifications.The identification was performed using morphological keys according to Minter et al. (1978), Woodward (2001), Tanaka et al. (2010), Luo et al. (2010) or morphological studies in Botella et al. (2010).All the isolates were examined from time to time and the rate of colony development was observed, as well as the fructifications in the culture, etc.The fungi were identified based on the sporulation in the culture, the appearance of hyphae, rate of growth, etc. Visual characteristics of symptomatic needles were examined with a stereomicroscope SZ51 (Olympus) and fungal structures observations were accomplished with a microscope BX41 (Olympus) under a 400× magnification.

Results and discussion
Asterosporium asterospermum (Pers.)S. Hughes, syn.Stilbospora asterosperma Pers. is fungus associated with a severe needle blight of ponderosa and lodgepole pines (Hunt, 1985) or scots pine (Kowalski and Kehr, 1992).In our experiments the fungus was isolated from needles of Austrian pine.Conidia stellate, brown composed with three arms were 15-17-25 × 10 µm, with two septa (Figure 1a).Number and length of arms are important distinquising criterion (Prášil and Réblová, 1995).Fruting bodies 1.5 mm in diameter were subepidermal at first, splitting irregularly to expose the dark spore mass later.Conidiogenous cells cylindrical, hyaline.Mature conidia massive, dark brown, smooth in pulvinate acervuli on twigs.Conidia appear as star shape that consisting of 4 arms at 90° to each other.The length of conidia was 20-55 µm from tip to tip of the branches which were 10-25 µm thick at the base.Conidia are formed solitary at the apex of coniodiophores (Boujari et al., 2012).According to Kobayashi and Kubano (1986) acervuli 800-930 µm in diam.occurred on dead beech bark are black, scattered, at first immersed within the epidermal layer, then erumpent.Conidiophores are slender, simple, hyalin to pale brown, 121.5-80 µm long.Dark brown, terminal, holoblastic conidia are smooth and consisting of 4 arms at 90° in angle to each other, connected with conidiophore at the center cell, 30-50 µm long from the tip of one arm to that of the opposite one: arms 15-25 × 7.5 × 12.5 µm with 3-4 septa.Asterosporium asterospermum isolated from Fagales trees with typical flattened acervuli with a wide opening.Conidiomata were pycnidial with a wide ostiole of more than 100 µm diameter.Conidial septation was euseptate.Central cell was indistinct.Conidia stellate, brown, composed of 4 equally developed arms, 30.5-55 (-61) µm between the widest points, arms 17-28 (-30) × 8-9.5 (-11) µm (Tanaka et al., 2010).
Fungus Camarosporium pini (Westend.)Sacc., syn.Hendersonia pini Westend., causes severe infection that can result in significant growth reduction.The fungus parasitizes the needles of Pinus nigra weakened by the low temperatures in winter period and by the drought from the spring to summer season.On the dry needles little black spots which represent pycnidia of 180-300 µm in diameter arranged linearly and in parallel to venation were observed.Pycnidia were immersed in the bark, at first scattered, than arranged in line, up to 700 µm diameter, subglobose, brown, perforating the epidermis, ostiole subpapilliform.Through the pycnidium pore there come out numerous oval brown conidia with three transversal walls and 1-2 vertical walls of sizes ranging between 18-20 × 9-10 µm.Conidia oblong, rounded at both ends, often slightly curved, at length 3-septate, not or hardly constricted, with frequently one or two longitudinal divisions, 15-18 × 7-8 µm, cells uniformely brown, the central cells often shorter than the terminal ones, sporophores short and indistinct (Grove, 1922).According Dennis (1964) spores elongate-oval, brown, 3-6-septate (or even 8-septate) and muriform, not constricted, 15-25 × 7-9 µm, sporophores indistinct.Comparable results with the frequent fungi in Camarosporium genus occuring on fallen cones on Austrian and Scots pine were obtained by Karadžić and Milijašević (2008) in Serbia.Cultures obtained during our cultivation from injured needles of Pinus nigra on PDA medium were initially white with abundant aerial mycelium, gradually becoming grey to dark grey.The reverse side of the colonies is at first white, but after 2-3 days becoming dark green to olive green from the centre.This coloration gradually spreads to the edge and becomes darker from the centre until the entire underside of the colony is black.Conidia were pale brown, thin-walled, with three transversal walls and 1, sometimes 2 vertical walls, smooth fusiform to fusiformelliptical, straight, apex subobtuse, base truncate 20-22 × 6-8 µm (Figures 1d, e).Results of comparable parameters achieved James (1984) on Pinus contorta.Spores were fusoid-elliptical, 4-celled, reddish brown and slightly constricted 15.5-22.0× 5.5-6.8µm in size.Pycnidia were immersed in host mesophyll tissue.Symptoms of damage on the lover crown side were observed.Over 20 species of Coleosporium cause rust diseases on pine.The first sign is small yellow spots in spring or early summer.Coleosporium asterum (Dietel) Syd.& P. Syd. is the best known of the group and infects 2-and 3-needle pines including Austrian, red, and Scote (Kunca and Leontovyč, 2013).The rust Coleosporium tussilaginis cause pine needle rust disease on Pinus sylvestris and Pinus nigra (Jones, 2006; Jurc, 2007).These needle rusts complete their life cycles in about one year and may begin on pine in later summer or early Autumn when it is infected by basidiospores produced on the alternate host.The fungus grows into and overwinters in the needles.Yellow spots develop on them the following spring.Fruiting bodies -spermagonia develop beneath these spots initially.Tongue-like fruiting bodies -aecia grow from these spots and when they burst, they release bright orange spores that will reinfect the alternate host (Pirone, 1978).In late summer develop telia on the margins of the uredinia (Nicholls et al., 1968; Nicholls andAnderson, 1978).Urediniospores in mass were first yellow orange, later white and in our experiments were subgloboid 18 µm, hyaline, with thick walls Close-up of urediniospores showing echinulate outer surface (Figure 1f ).According to Gjaerum et al. (2008) individual urediniospores were subgloboid or ellipsoid, 18-28 × 16-22 µm, walls 1 µm thick, hyaline, and densely verrucose.Basidiospores which develop from the telia, will infect pine needles.A few species of Coleosporium will survive for more than one year as mycelium in the living tissue of the pine host (Karadžić and Milijašević, 2008).
Lophodermium needle cast disease is caused by the fungus Lophodermium seditiosum Minter, Staley & Millar which infects needles of Austrian (Pinus nigra), mugo (P.mugo), red pine (P.resinosa) and Scotch pine (P.sylvestris) and can kill young trees within a year (Karadžič and Milijaševič, 2008;Rajkovic et al., 2013).This fungus overwinters as vegetative mycelium in needles infected the previous season.Fungal fruiting pustules develop on fallen needles over the summer.Windborne spores during wet periods (from August to October) are released, which infect the current year's needles, resulting in a small brown spot, often with a yellow halo.The spots on needle later coalesced to give blighter appearance and resulted in premature defoliation.Reproductive structures of the fungus can often be seen on dead needles.On native pines needlecast is most evident in late spring to early summer when needles shen.Examination of blighter needles showed the presence of small pycnidia which erupt through the epidermis of infected needles.Conidia were 8.34 × 1.60 µm in size (Ahanger et al., 2011).In our experiments this fungus on 2% PDA fast growing and formed small pycnidia with rod-shaped, small conidia about 7-8 × 1 µm in size (Figrue 1h).According to Minter et al. (1978), cultures of Lophodermium seditiosum on 2% malt agar initially fast-growing.Growth invariably terminating before the edge of the Petri dish is reached.The fungus formed pycnidial, subepidermal, often coalescing, 300-500 µm long conidiomata, and bacillar, 6-8 µm long conidia.Similar results achieved Rajkovic et al. (2013) by experiments with fungus Lophodermium seditiosum on some pine trees includes Austrian pine.Conidia 7×0.7 µm were smaller, rod-shaped, cylindrical, ends rounded, straight or slightly curved to a side, hyaline and aseptate.
Fungus Phomopsis causing cankers, needle loss, branch death and often tree death of mature trees.Phomopsis shoot blight causes young needles and shoots to turn brown and curl downward.Symptoms of Phomopsis leaf spot are small irregular, or round shaped, pale green-to-yellow spots with dark centers.Dark irregular shaped lesions develop on infected shoots (Nita et al., 2003;Karadžič and Milijaševič, 2008).Strees from environmental conditions have played a role in the development of Phomopsis decline.Moisture is an important factor in disease spread and severity.Small black fruiting bodies -pycnidia with two types of conidia: alpha and beta are formed on this dead tissue (Woodward, 2001).α-conidia are hyaline, nonseptate oblongelliptic in shape with a size of 6.3-11.2× 1.7-2.8µm, and β-conidia are hyaline, nonseptate, filiform and curved in shape with a size of 20-25 × 1 µm (Nita et al., 2003).According to some literature data size of spores occurs from 5 to 18 × 2-4 µm, α-conidia often 7-10 × 2.5 µm.In our experiment with Pinus nigra needles we obtained formation only α-conidia, which are fusiform, 10 × 2-2.5 µm.On both tapering edges we occurr one oil drop (Figure 1j).Conidia are produced in these structures and when moisture is present masses of conidia will ooze out to form tendrils or horns.The conidia are spread by rain and various other means such as mechanical disruption and insects.The fungus enters the plant through wounds as well as unbroken tissue.The browning of the needles and twigs begins at the tips and progresses downward toward the stem.
Trichothecium roseum (Pers.)Link syn.Cephalothecium roseum Corda, Sphaeria rosea Pers., Trichoderma roseum Pers. is a filamentous mitosporic fungus word-widely distributed, which reduced germination.The fungus is mostly saprophytic or weakly parasitic (Barnett and Hunter, 1972).It was found as laboratory contaminant, was previously recorded on felled trunks and fallen branches of Acer, Corylus, Fagus, Prunus, Quercus and Ulmus (Ellis and Ellis, 1985), or of many conifers (Mason and Arsdel, 1978;Karadžič and Milijaševič, 2008).Our survey has resulted in finding that T. roseum is the rare pathogenic fungus isolated from inspected leaf tissues in connection with attacked cherry laurel trees (Bernadovičová and Ivanová, 2011).Colonies of Trichothecium on PDA mediu grow rapidly, are flat, granular and powdery.From the front, the colour is white initially and becomes pale pink to peach-coloured, reverse is pale.The conidiophores are more or less rough-walled, long, erect and unbranched.Conidia in our experiments are bicellular, smooth, elliptical or pear form with one septum, each with a flattened protuberance at the base, hyaline or bright, slightly thick-walled, formed an elongated clusters at apex of conidiophore, upper cell usually langer than basal cell, 16-20 × 5-7 µm in size (Figures 1k, m).Biometric characteristics of asexual spores all obtained fungi are in Table 1.Comparison of biometric characteristics and morphological features of other fungi also isolated and determinated before on genus Pinus sp. are reported in Ivanová andBernadovičová (2010), Pastirčáková et al. (2014), Ivanová and Ondrušková (in press).

Acnowledgements
This study was carried out with support from the scientific projects VEGA of the Slovak Academy of Sciences No 2/0069/14 and 2/0071/14.