Winter desiccation of dwarf pine ( Pinus mugo Turra) needles in the area of Belmeken Dam

In the summer of 2018, browning and drying of the needles on the dwarf pine ( Pinus mugo Turra) formations in the vicinity of the dam Belmeken (Rila Mt.) were found. The damages were concentrated mainly in the southern part of the plants and affect the young needles on the tops of the shoots. During the route survey conducted in 2019, almost complete recovery of the affected plants was observed. Symptoms of drying were found only on single bushes. Studies and laboratory analyses gave reason to conclude, that the drying was due to adverse weather conditions in the winter of 2017-2018, when there was significantly less than normal rainfall, and sudden temperature changes and extremes were found. Established in the dry needles, fungi of the genera Lophodermium, Hendersonia and Neofusicoccum can be characterized as weak parasites, manifested secondarily, or as endophytes, which under physiological stress due to changes in certain environmental factors can become latent pathogens, worsening the condition of dwarf pine.


Introduction
Drying, also known as leaf blight, is a physiological problem associated with water supply disorders. The reason for leaf blight may be the lack of water in the soil to supply the roots during periods of drought in summer or in winter when the water in the soil is frozen, but it can also be caused by a pathogen that develops in xylem. Summer leaf blight is a problem mainly for deciduous species. Winter drying (burns) is most often associated with conifers (Bencheva, 2017). W arm, windy and sunny weather during the winter months accelerates transpiration, while the water in the soil may be frozen and indigestible. The damage is due to a sudden change in temperature, which usually occurs at sunset and sunrise. The southern side of the tree, exposed to the sun, is more often damaged. In evergreen conifers, the symptoms of burns appear as necrosis from the tip of the needle down in late winter or early spring, but may not appear until summer. They are easily recognizable by the browning of the needles and drying above the snow level. The part of the tree under the snow is usually not affected. Trees on the periphery of forests and young plants are more susceptible.
During winter drying, the bases of the needles and some whole needles often remain green. In most cases, the damage is insignificant and the trees are restored. Buds at the tops of the branches usually remain viable and the tree can stimulate new growth. However, sometimes the buds, even the affected branches, can be killed, the growth in height and diameter of the damaged trees may lag behind and they are prone to attacks by other organisms. These are Phacidium infestans P. Karst., Lophodermella sulcigena (Link) Höhn., Gremmeniella abietina (Lagerb.) M. Morelet, that cause great damage to pine plantations in mountainous areas of Serbia (Karadžić, Milijašević, 2008), Mycosphaerella dearnessii M.E. Barr and Coleosporium tussilaginis (Pers.) Lév., found in Poland on dwarf pine (Pusz, Kita, 2014).
The reason for this study is to establish the complex of pathogens that caused mass browning and drying of dwarf pine (Pinus mugo Turra) needles in the region of Belmeken in the summer of 2018 (Fig. 1).

Materials and methods
Two route surveys were conducted in the autumn of 2018 (on October 1 and November 2) in the vicinity of the Belmeken dam, located at 1923 m above sea level in the Rila Mountains. The survey site has geographical coordinates 42˚08'55.00"N; 23˚47'03.44"E. It was found that signs and spore-bearing structures specific to some phytopathogens were observed on individual brownish needles of many of the plants in the dwarf pine formations, therefore samples were collected for identification in the Laboratory of Forest Phytopathology of the University of Forestry, Sofia. Twenty plant samples were microscopically examined. A minimum of 50 spores were measured from the different spore formations identified.
Another route survey of the same area was conducted in the summer of 2019 to monitor the development of symptoms.

Results and Discussion
During the route surveys conducted in 2018 an unevenly distributed drying of the needles was found on the dwarf pine formations. The lesions are concentrated mainly in the southern part of the plants and affect the young needles on the tops of the shoots.
Microscopic analysis of the collected symptomatic plant samples showed the presence of spores of various fungi.
Based on these data, it can be concluded that the fungus we found is Hendersonia pinicola (Phaeosphaeriaceae, Ascomycota). Hendersonia pinicola has been described mainly as a secondary pest following previous attacks by Lophodermella sulcigena (Link) Höhn. -in North America on Pinus contorta (James, 1984), Pinus banksiana Lamb. and Pinus strobus L. (Durand , and in Europe on Pinus sylvestris L., P. mugo, P. nigra, P. contorta (Čahtarević, 2013). It is believed that the action of Hendersonia spp. can be considered as a natural form of biological control. Studies by Alkanen (1985) in Finland on the relationship between Lophodermella sulcigena and Hendersonia acicola show that the antagonistic effect of H. acicola against L. sulcigena occurs only when H. acicola infects needles in autumn, but not in spring.
In Europe, Lophodermella sulcigena causes Pinus mugo disease , which is manifested by redness on the tips of young needles of the current year, which later turn yellow-brown and finally gray. Elongated elliptical black apothecia are located on the killed parts of the needles, below the epidermis, without lips and transverse stromatic lines along the needles. It does not infect all trees in the plantations. Pinus sylvestris, P. nigra var. maritima, P. contorta are also hosts. Lophodermella conjuncta (Darker) Darker causes a similar change in color, but symptoms develop on biennial needles (Mitchell et al., 1978). The asci of L. sulcigena are unitunicate, curved, with 4 or 8 spores, 110-140 × 13-15 µm (100-160 × 15-16 µm at L. conjuncta). Its ascospores are 27-40 × 4-5 µm in size, transparent, curved, not septated with a smooth surface, wrapped in a mucous membrane. They are spread by wind in humid weather. Ascospores in L. conjuncta are more elongated (75-90 × 3-3.5 µm).
L. sulcigena has been reported in Bulgaria (Dobreva et al., 2016), but there are no data on morphological and DNA analyses. In Serbia, it has been found to attack young, underdeveloped needles from the current growing season (Karadžić, Milijašević, 2008). Infection occurs at the base of the needle, with the part above the site of infection dying and changing color, becoming grayish or light brown, while the part below the site of infection remains green. The gradual killing of the tissues continues until the beginning of November, and only one of a pair of needles may die. The infection rate is highest in years with heavy rainfall in May and June. This fungus is found mainly in mountainous areas, as the ecological conditions at lower altitudes are not favorable for it.
Based on the size of ascuses and ascospores, the fungus found by us differs significantly from Lophodermella sulcigena and L. conjuncta, as well as from Lophodermium pini-mugonis and L. seditiosum. Lophodermium corconticum is close to the size of the ascus, but the ascospores are shorter. To the greatest extent, our sizes are close to those of L. pinastri. The latter two species form sporocarps mainly on dead fallen needles as L. pinastri develops on 2-year-old needles.
From our known information about the development of the described species of fungi and their morphological data, it can be assumed that the fungus found on dwarf pine in Belmeken is Lophodermium corconticum (Rhytismataceae, Ascomycota), but its exact identification is not possible without molecular analysis.
Neofusicoccum parvum is distributed worldwide on a large number of hosts (from families Anacardiaceae, Cupressaceae, Ebenaceae, Fagaceae, Juglandaceae, Lauraceae, Moraceae, Myrtaceae, Oleaceae, Pinaceae, Proteaceae, Rosaceae, Rutaceae, Vitaceae). Phylogenetically, this species is in a group of morphologically similar species that can be distinguished only on the basis of data from molecular analyses.
Most members of the Botryosphaeriaceae family have been described as endophytes and latent pathogens (Slippers, Wingfield, 2007), incl. the genera Neofusicoccum and Diplodia. The development of disease in them is associated with some form of stress (drought, hail, damage from other pathogens, insects, snow, frost) or deteriorating growth conditions for trees.
Zlatkovic et al. (2019) study the genetic diversity of the populations of N. parvum and D. sapinea from the Western Balkans (Serbia and Montenegro), where damage by members of the Botryosphaeriaceae family is increasingly found. The low genetic diversity and dominance of N. parvum and D. sapinea on non-native trees leads them to conclude that these species were most likely introduced to the Western Balkans with infected plants (Botryosphaeriaceae endophytic infections are asymptomatic and can easily go unnoticed).
During the 2019 route survey of dwarf pine formations in the area of Belmeken Dam, almost complete recovery of the affected plants was found. Symptoms of dry- ing have been found in single bushes. This fact, as well as the uneven distribution of drying lesions found in the previous year, concentrated mainly on the southern side of the plants and affecting the young needles on the tops of the shoots, both give us reason to look for abiotic causes of the phenomenon.
Similar symptoms have been found in different parts of the world. In some North American states, the needles of many pines, spruces and other conifers turn brown during the winter. This is often observed on ly in the upper parts of the tree, if the lower branches are under the snow cover, protecting them from wind and sun (Williams, 2018). In Colorado, a sharp drop in temperature in 24 hours by more than 10 °C in November 2014 caused winter drying, especially in the area of dwarf pine and Austrian pine introduction (Ciesla, 2015). The warm weather slows down the hardening of the shoots so far. Damage is most severe in exposed parts of the trees and is observed in late winter and early spring. The side of the tree that faces the prevailing winds, usually from the west, is most susceptible to winter drying.
The reference made for the monthly temperatures and precipitation in the winter of 2017-2018 (Table 1) at the nearest meteorological station to the Belmeken dam on Musala peak (www.stringmeteo.com) shows a tendency of increase in the average monthly temperatures by 0.1-2.8 °С and a reduction in the monthly precipitation amounts to 31-90% of the average monthly norms for a 30-year period . In addition, sharp changes (extremes) in daily temperatures were reported every month, which, for example, in December 2017 for 12 days consistently decreased and increased by more than 15 °С. These conditions are suitable for the occurrence of winter drying or the so-called air embolism. At high altitudes, winter weather conditions often cause severe stress, which can cause tree embolism (Maruta et al., 2020). The phenomenon of air embolism in plants occurs in the xylem when the water pressure becomes so high that the air there expands, blocks the vessels with an air balloon and clogging (embolization) of the vessel occurs. This reduces the plant's ability to transfer water from the soil to the leaves. In Japan, Maruta et al. (2020) investigated the onset and recovery from winter embolism in Abies veitchii growing near the upper forest boundary. They found a complete (100%) loss of xylem conductivity on the windward side of the trees and 40% on the leeward side. The recovery of conductivity in the xylem on the leeward side begins gradually at the end of winter, while on the windward side it occurs in June. Winter embolism and recovery were also observed in Pinus mugo in Austria (Mayr et al., 2019).

Conclusion
The performed researches and laboratory analyses give grounds to conclude that the drying in the dwarf pine formations established in 2018 in the area of Belmeken Dam was caused by unfavorable weather conditions in the winter of 2017-2018, when significantly less than normal rainfall fell and sharp temperature changes and extremes were found.
Future changes in available snow cover may have a strong effect on Pinus mugo and other shrubby plants, as reduced snow cover exposes plants to increased temperature stress and impairs recovery processes.
Established in the dry needles fungi of the genera Lophodermium, Hendersonia and Neofusicoccum can be characterized as weak parasites, manifested secondarily, or as endophytes, which under physiological stress due to changes in certain environmental factors can become latent pathogens, worsening the condition of dwarf pine.