Review of the most important pathogens in Serbian forest nurseries

Successful reforestation depends on the quality and health of seedlings. Targeted production in forest nurseries should produce plants that will be able to survive unfavorable environmental and weather conditions in Serbia. Pathogens can reduce the vitality of seedlings and decrease survival after outplanting. The most common pathogens identified on seeds of both conifer and broadleaved seedlings are presented. The most frequently used fungicides for the control of pathogens in Serbian forest nurseries, as well as the best time of their application, are reviewed.


Introduction
Serbia, having 29.1% (2.252.000ha) of its area covered in forest, is one of the mild-forested countries of Europe (Banković et al. 2009).The forestry sector is currently under pressure to improve the situation and increase reforestation efforts.An action plan for Serbian forestry has predicted an increase of forest coverage to 35% by 2050.The reforestation of such a large area will pose many challenges to the forestry professionals; among them, seedling health will certainly be one the most challenging.
Forest ecosystems throughout Serbia consist of 49 autochthonous woody species.Broadleaf species (40) dominate over coniferous species (9) (NFI 2009).Despite the great diversity of trees, only a few species dominate in Serbian nursery production.

Main groups of pathogens in forest nurseries
Intensive production of seedlings depends on a constant supply of seeds.To ensure the quality of descendant seedlings, seeds are collected in established seed orchards and selected stands (Ivetić and Vilotić 2014).Collection and manipulation of seeds present many opportunities for contamination by various microorganisms (viruses, bacteria, phytoplasma, fungi, etc.) (Salisbury 1953).They can cause decay and destroy seed nutritional reserves or act as saprotrophs that cause no damage to the seed or seedling (Peno 1971).
The first step to assess the presence of microorganisms consists in routine seed tests for germination and purity.Sometimes a seed may appear to be healthy, even if it is abundantly infected with pathogens (Rees and Phillips 1986).Laboratory tests will not detect some pathogens that can cause damage in the field, on the germ tube, or to seedlings.A ten-year study of mycobiota on four coniferous and the five most important broadleaved hosts is presented in Table 1.
Among studied samples, the most frequently observed species were from the Penicillium, Aspergillus, and Fusarium genera and Botrytis cinerea.Some species from the genus Fusarium can cause damping-off on young seedlings not only during the first few weeks after germination, but also later, on seedlings up to the age of one year.The most-frequently observed species are not host specific and can destroy the seed of many tree species (Sutherland et al. 2002).

Seed-borne diseases
Aspergillus species are well known contaminants of various substrata and are common seed contaminants.A considerable number of species (A. clavatus, A. fumigatus, A. nidulans, A. niger, etc.) can be found on seeds, especially if seeds have been stored in humid conditions and have reduced germination capacity (Malone and Muskett 1964).Production of various chemical substances, such as alcohols, enzymes, antibiotics, and toxins, can cause diseases in plants, animals, and man (Bennett 2010).The species that could be linked to seed damage is A. niger, which was also reported to have caused systemic infection on ornamental plants (Samson et al. 2001).
Penicillium species are known to contaminate bulbs, rhizomes, and fruit, but are generally considered as saprophytes (Bennett 2010).Sometimes the secondary spore contamination of seeds is quite large, but it is generally less than 1%, particularly in laboratory conditions in which germ tubes were infected with representatives of this genus.Trichothecium roseum Link ex Fr. is a saprophyte of various substrates, but in some conditions it could act as a weak parasite (Malone and Muskett 1964).Browning of the fruit (a nut-like drupe) and cotyledons of Tilia species during seed health tests was observed.The cause of this phenomenon, however, was not thoroughly studied.
Phomopsis spp. was associated with acorns of Quercus spp.and other tree species without any further information on pathogenic behavior (Kehr and Schroeder 1996).Both pathogenic P. quercella and saprobic P. glandicola were observed on Q. robur and Q. petraea acorns.In this study, Phomopsis was found to abundantly spoilage non-germinating ash seeds (Fraxinus spp.), but we could not confirm that the species was the cause of seed death.The Rhizopus genus has many species that are regularly found on seeds.Samples from coniferous hosts originating in the western regions of Serbia were severely contaminated with Rhizopus spp.
Graphium, similar to Acremonium and Aureobasidium, was rarely observed and was without obvious influence on the cotyledons and the seed germ tube development.Some Acremonium species (e.g. A. implicatum) are entophytic and are transmitted in the wild by seeds of Lolium spp.(Ewis et al. 1997).
Many molds, including Mucor mucedo de Bary and Woron., are considered as saprophytes that are present in the laboratory; contamination of seed lots is inevitable during routine tests.
Observed bacteria contamination was significant for some examined samples, but the destruction of seeds and cotyledons could not be correlated to the bacterial infection.+++ strong pathogens that could be the practical problem; ?? unknown trophic behavior (Keča et al. 2015).

Conifer tree diseases
Conifers are the dominant species produced in forest nurseries in Serbia.Generally, conifers are susceptible to the attack of pathogens in all phases of development, from germination, in the seedling phase, but also during growth in the second and third year.A wide range of diseases can cause problems on root systems, stem and needles.The most often observed in nurseries are presented.
Chrysomyxa pirolata Wint. is a pathogen that can frequently be found on spruce cones and cause their destruction.It was earlier reported that C. pirolata decreases the quality of Norway spruce seeds (Lilja et al. 2010).According to Karadžić (2007) up to 90% of seeds from infected cones show a decrease in weight and vitality.
Fusarium is a genus with a great number of plant-pathogenic species, ranging from 53 (Lević 2008) up to 1,000.These cause diseases in several agriculturally important crops, including cereals, and could also be harmful to humans and animals (Booth 1971).Different Fusarium species can be found on seeds (F.proliferatum (Matsush.)Nirenberg ex Gerlach and Nirenberg, F. semitectum Berkeley and Ravenel, F. subglutinans (Wollen et Reink) Nelson, Toussoun & Marasas (Cvjetković 2011), but most post-emergence damping off in pine production was related to F. oxysporum or to F. oxysporum subsp.pini and F. avenaceum (Fr.)Sacc.(Lilja et al. 1995).Damage is usually present if soil humidity is too high or after late sowing, when temperatures reach 22-26°C (Karadžić and Anđelić 2001), which is optimal for development of fusariosis (Sutherland and Davis 1991).Soil pH (6-7) and humidity are important for Fusarium incidence; fertilization can accelerate the infection process.Less damage is observed in sandy soil as well as rich soil with antagonistic organisms.
Rhizoctonia species damage is more prevalent in natural regeneration than in nurseries.Inadequately treated seeds, especially those of beech or seedbeds, can exhibit substantial losses due to Rhizoctonia solani Kühn, while damage is rarely observed in container production.Among resistant conifers are Juniperus, Cupressus, Thuja, and Chamaecyparis, while Quercus, Ulmus and Robinia are considered as resistant hardwood species (Tomić et al. 2011).
Grey mold can be found as a saprophyte in almost all nurseries in its teleomorphic (Botryotinia fuckeliana (de Bary) Whetzel) and asexual stages (Botrytis cinerea).The fungus is usually present on dead plant parts or lower branches (Lilja et al. 2010).The first symptoms of its presence involve needle chlorosis, with later developments of infection, greyish mycelium and the presence of branched conidiophores that appear on necrotic tissues (Lilja et al. 2010).Damage from this parasite is connected with other abiotic damage such as frost, herbicides, low light intensity, extended drought, or water saturation (Zhang and Sutton 1994).In the Balkan region, seedling decline is also usually connected with cold and wet weather (Karadžić 2010).Under these conditions, even healthy plants could be infected (Karadžić and Anđelić 2001).It was observed that two-year-old seedlings could die in less than three weeks under those conditions.Weedy nurseries with dense seedbeds that maintain high relative humidity favor pathogen infections.Forest nurseries that are closer to the plantations on natural forests are exposed to the inoculum produced in forest litter.Spores germinate between -2 and 32°C (Karadžić and Anđelić 2001) with the optimal temperature for infection being 7-25°C (Petäistö 2006;Karadžić and Anđelić 2001).Spores germinate and infections occur within three hours if water is present on the plants surface.Keeping the canopy well aerated and as dry as possible can reduce infection (Mittal et al. 1987).Irrigation should occur during the morning hours to enable the faster drying of needles; also reduction of nitrogen could prevent incidences of grey mold.Spruces (Picea spp.) are sensitive to Botrytis cinerea attack, but the pathogen can also be found on larch (Larix deciduas Mill.), Douglas fir (Pseudotsuga menziesii (Mirb.)Franco), sequoia (Sequoiadendron giganteum (Lindl.)J.Buchh.), and cypress (Cupressus spp.) seedlings (Williamson et al. 2007).It was observed that Serbian spruce (Picea omorika (Pančić) Purk) could suffer very severe damage if not grown under adequate conditions.Botrytis spp.may develop tolerance to fungicides that are used repeatedly, so fungicides should be used in rotation during the growing season.
Phytophthora and Pythium species are well-known pathogens of seedlings and young trees in nurseries and plantations.During the 20 th century, the most damage in nurseries was attributed to P. cactorum (syn.P. omnivora) (Karadžić and Anđelić 2001).Recent results show that at least 15 species are present in Serbian forest ecosystems, plantations, and nurseries (Milenković 2015).Phytophthora can cause pre-emergence decay of the germ tube, damping-off, and root rot in 1-2 year old seedlings.Because of motile reproductive structures, the presence of free water in poorly drained soils increases the chance of damage from both species.Because resting oospores in dead plant parts also play an important role in disease control, reduction of inoculum potential is important.
Spruce needle rust (Chrysomyxa abietis (Wallr.)Unger) is a pathogen native to Europe and Northern Asia.This rust does not require an alternative host.Hyphae penetrate through the stomata and cause chlorosis after several weeks (Hansen 1997;Uotila et al. 2000).Basidiospores are released during wet periods in May and June.Needles fall off after the basidiospores are released.Picea abies (L.) Karst, Picea sitchensis (Bong.)Carrière, Picea rubens Sarg., Picea pungens Engelm., and Picea engelmannii Parry ex Engelm.could be hosts for C. abietis (Uotila et al. 2000).Diseased needles are observed in Serbian nurseries mostly on Picea pungens.Control should be applied to severely infected plants from the end of April until June; older infected trees in the vicinity of nurseries should be cut down (Karadžić and Milijašević 2003).
Needle cast diseases are caused by Lophodermium species, the most important pathogen of pines in nursery production (Lazarev 2006).Evidently, no resistant pine species exist, but the damage to Scots pine is the most important (Lilja et al. 2010).Lophodermium seditiosum Minter, Staley and Millar attack needles from current vegetation, while L. pinastri (Schrad.)Chevall.infects needles from the previous year.Damage can be also seen on Austrian and Aleppo (Pinus halepensis Miller) pines.The critical period for needle infections occurs from mid-August to the end of September.The first symptoms can be observed 2-3 months upon being infected (November-December) and depend on environmental conditions.Infected needles first turn red, then brown, and fall off the following spring (Lazarev 1981).Higher precipitation during autumn favors ascosporic infection (Lilja et al. 2010).If planting is done in the early autumn, seedlings can appear healthy, but disease development could worsen their condition during winter and kill them by the following spring.Early identification of latent infections is crucial because infected seedlings do not survive planting stress (Lilja 1986).Seedling protection is necessary for the production of healthy material.Annual application of fungicides has become a standard process in most European countries (Lilja et al. 2010;Lazarev 2006).Two treatments, at the end of August and in mid-September, are sufficient if copper-based fungicides are used.According to the Rule Book on Health Inspection of Planting Material in Nurseries, up to 10% of seedlings could have needle cast infection.An infection of less than 5% of a plant's needles is tolerated (Official Gazette of R. Serbia).
Teleomorph is rare and perithecia are produced on 2-3 year-old needles.Serbia is among a few countries where teleomorph is found.Infections occur during May and June; red -band symptoms can be seen from late September and become very clear in November and December (Karadžić 2004).Austrian pine is very susceptible to the attack of Dothistroma needle blight, while other economically important pine species show moderate or low susceptibility (Karadžić 2004).The situations in Central and Northern Europe are somewhat different because the most of the DNB damage is observed on Scots pine.Control in nurseries is necessary and consists of applications of copper-based fungicides during the critical period of infections (May-June).
Snow blights are usually observed in nurseries established on higher altitudes where snow covers seedlings at an earlier time.Symptoms become visible after the snow melts in spring.Phacidium infestans P.Karst.and Herpotrichia juniperi (Duby) Petrak attack needles and develop mycelium that grow under the snow.A whitish or blackish mycelium covers needles and prevents them from falling off.It was observed in Finland that P. infestans could attack container seedlings of Norway spruce (Lilja et al. 2010).In the Balkan region, snow blights are quite often observed on Norway and Serbian spruce, Scots pine, and rarely on European fir (Abies alba Mill.), Junipers (Juniperus spp.), and Thuja spp.(Karadžić and Anđelić 2001).Infections occur in October and November before the first snow of the season falls.Ascospores are released at temperatures close to 0°C (Kurkela 1995).Further development of the disease depends on the height of snow.P. infestans is a quarantine disease and should be prevented from migrating to new regions (Karadžić 2010).Control in nurseries and plantations are possible by spraying fungicides as late as possible (Lilja et al. 2010;Karadžić and Anđelić 2001).
In nurseries, Diplodia sapinea (Fr.)Fuckel (syn.Sphaeropsis sapinea) is generally considered as a wound parasite, infecting tissue that has already begun to wilt due to other causes, such as drought or wrench, during dry weather (Milijašević 2003).Diseases seldom progress to cause the death of the seedling, but the destruction of leading shoots renders the plant inadequate for planting.Nurseries should be established in the vicinity of pine plantations.Effective control could be achieved by spraying with fungicides from mid-April until mid-June (Milijašević 2002).

Broadleaved tree diseases
It is a common opinion that pathogens cause less damage in broadleaved nurseries in comparison to conifers.However, some diseases are observed quite regularly and can cause significant loss in number and reduce seedling quality from year to year.
Erysiphe alphitoides Griffon and Maublanc (syn.Microsphaera alphitoides Griffon and Maublanc) is a serious disease of oak species (Quercus spp.).The disease appeared as an epidemic at the beginning of the 20 th century in France, from where it spread throughout Europe.Damage could be observed in nurseries, plantations, and natural stands.Erysiphe species attack young leaves and soft shoots, covering them with whitish mycelium.Powdery mildew can have a damaging impact on natural oak regeneration and a significant role in the decline of mature trees (Marcais and Desprez-Loustau 2014).Affected seedlings have reduced height increments of as much as 30% (Karadžić and Anđelić 2001).Ascospores are released during April and May from chasmothecia and overwinter on fallen oak leaves.Conidia are produced on infected leaves from mycelium during the vegetation period, while young flag shoots become infected by mycelium that overwinter in buds.After the first growing season, wellformed oak plants 2-3 years of age, tolerate the presence of powdery mildew which is no longer a limiting factor for their development in natural regeneration (Pap et al. 2012).Systemic fungicides, applied from June to the end of August are efficient and necessary in the control of oak mildew (Glavaš 2011;Karadžić and Anđelić 2001).
Cryptodiaporthe populea (Sacc.)Butin (anamorph Dothichiza populea) causes canker on poplar trees, spreading widely in nurseries and hybrid poplar plantations (Keča 2008;Ostry et al. 2014).Damage is most often observed on the representatives of section Aigeiros.On older trees in natural stands and plantations, infections usually occur on branches with formation of cankers; on cuttings in nurseries, the main stem becomes infected.The main inoculum consists of conidia that are produced, in picnidia, on the edges of a necrosis and spread within drops of water (Marinković 1965).Infections proceed through lenticels, buds, injuries, and the base of the shoot.Nonsuberized leaf scars from early defoliation, caused by rust or Marssonina species, could serve as an entrance for Dothichiza (Tomović 1990).Most infections occur in late autumn, late winter, and early spring before vegetation starts.Control should be based on disease prevention by keeping plants in optimal growth conditions.Nurseries should be established on soils with a good texture and structure for poplar growth.Irrigation, tillage, use of herbicides to remove weed competition, and protection from leaf diseases, will avoid predisposition to Dothichiza and therefore are efficient in disease control.Fungicides are necessary for the production of healthy nursery material.
Poplar rust forms orange urediniospores on the under or upper side of leaves in July.Melampsora allii-populina and M. larici-populina produce uredinial and telial stages on the Poplar spp.leaves, while the aecial stage has been observed on Allium leaves and Larix spp.needles (Keča 2008).Aecidiospores transfer infection to poplars during summer when damage has been observed on leaves.Infection is followed by the production of pustules and necrotic lesions at infected sites.Differences in susceptibility among cultivars have been reported (Keča 2003b).Susceptible clones loose leaves quite early (in late August).The development of a disease is, beside differences in clone susceptibility, greatly influenced by weather conditions.It was shown that P. x euramericana cv."Pannonia" is more resistant to attack than other clones grown in Serbia.Disease control can be obtained by strict hygiene in nurseries (Lilja et al. 2010).The application of fungicides labeled for control of Melampsora on poplar from June until mid-July reduce infection levels on leaves (Ostry et al. 2014).
Marssonina leaf spot (Drepanopeziza punctiformis Gremmen) is one the most important foliage diseases of Populus species (Beare et al. 1999;Keča 2003aKeča , 2003b;;Keča et al. 2015).After infection of ascospores, small (up to about 1 mm in diameter) circular-to-irregular dark-brown spots develop on leaves during spring.Necroses spread on leaves and cause premature defoliation, weakening, and dieback of infected trees.Fast-growing hybrid poplars that were planted worldwide after the Second World War are now ready for the production of technical wood and woody biomass (Keča et al. 2012;FAO 2011).Marssonina and other leaf spot causing pathogens (Septoria, Phyllosticta, etc.) considerably reduce the growth and productivity of hybrid poplars and cause significant economic loss (Zhu et al. 2012;Keča 2008).The most promising control is to select and grow poplar varieties resistant or tolerant to the disease (Zhu et al. 2012;Han et al. 2000).Application of fungicides during the summer months is necessary in nurseries in order to reduce leaf infections and prevent early defoliation (Keča 2003b).
Further development of marker-assisted and biotechnological approaches in breeding and selection of resistant poplar clones are currently limited, but will play an important role in the future.

Disease prevention and control measures
The application of control measures is in Serbian nurseries usually connected with the occurrence of the pathogens or damages.A treatments' effectiveness is dependent on correlation between timing application measures, and the host and pathogen phenology.There are still no developed plans for protection against the most important diseases, so procedures depend only on the personal involvement of nursery personnel/managers responsible for the production.Chemical control measures options for the most important forest nursery pathogens, and the best time period for application are presented in Table 2.
The application of chemicals for diseases control is quite questionable in forest nursery production in Serbia.Available fungicides are registered mainly for agricultural crops.Wholesalers do not find interest to register for the use in forestry because this market is very small.

Concluding remarks
Intensive production, as in nurseries, presents a constant risk of damage caused by pests and diseases.Factors such as high seedling density, irrigation, fertilization, and herbicides, encourage the development of pathogenic fungi.Hygiene in nurseries and application of available measures in seed management, disinfection of soil, and treatments by pre-emergence and post-emergence pesticides prevent the survival of pathogens and the development of diseases.Application of pesticides at a critical period for infections maximizes their efficiency and minimizes environmental contamination.
Educating nursery employees about pathogen life cycles and ecology is necessary for symptom recognition and the application of appropriate protective measures.Modern technologies can help identify diseased seedlings and thereby facilitate their exclusion for their use in reforestation.Recent changes in climate require the use of vigorous and healthy plants that will be able to survive environmental extremes.

Table 1 .
Nursery pathogens observed on seeds and seedlings in Serbia

Table 1 .
Nursery pathogens observed on seeds and seedlings in Serbia, cont.

Table 2 .
Chemical protection periods and fungicides used in the forest nurseries in Serbia.