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Науковий вкппк НЛТУ Украши. - 2015. - Вип. 25.8
growth within the limits of the selected exposition-orographic groups (EOG) and in type site conditions (TSC) of C2-C3 and in to TSC of D2-D3. The equalizations coefficients are determined with the proper preciseness. Forest stands of Ib stand quality class of both groups of TSC on I EOG characterize silver fir forest stands the maximal values of forest stands height. Some features of dynamics of relative stocking are analogical - the maximal values of index are marked similarly for forests stands of Ib stand quality class. Maximal values of other assessments indexes of probed forests stands of I and II EOG in to TSC of C2-C3 are similarly marked in forest stands of Ia stand quality class, and in to TSC of D2-D3 - in forest stands of I stand quality class.
Keywords: mountain modal silver fir forest stands, exposition-orographic groups, assessments indexes, dynamics.
УДК630*232.3 Eng. N.I. Hrunyk; researcher Yu.M. Yusypovych, PhD;
senior researcher VA. Kovaleva, PhD; prof. R. T. Gout, Dr. Sci. -Ukrainian National Forestry University, Lviv
HETEROBASIDION ANNOSUM ROOT ROT INFECTION DEVELOPMENT IN SCOTS PINE AND EVALUATION OF THE EXPRESSION LEVELS OF LIPID TRANSFER PROTEIN AND DEFENSINS IN INFECTED TISSUES
Heterobasidion annosum is a causative agent of the root and butt rot and appears to be one of the most economically important conifer pathogens, which causes havoc in boreal forests at northern hemisphere. Despite biology and genetics of this fungus is well studied, the mechanisms of defense responses and resistance of pine trees are still unclear. Recent advances in transcript profiling and molecular characterization of pathogenicity factors approached us in our understanding of this system. In this study we showed features of the development of pathological process in Scots pine saplings caused with annosum root rot at different periods of their growth and changes in expression levels of a lipid transfer protein and defensins by semi QRT-PCR
Keywords: Scots pine, defensin, lipid transfer protein, expression, annosum root rot
Introduction. Conifer trees in Ukraine make up 23 % of total forest area, and in Polissya that area is 64.5 % making them very important tree species in Ukrainian forestry [1]. Maintenance of healthy stands is a guarantee of quality forest preservation, but in nature is impossible to reach such state, as a lot of factors are contributed to the forests formation, which positively or either negatively affect the stands quality. Among those factors the disease management is one that can be improved by a human. Thus development of the effective methods of disease management is very important by mean to prevent significant losses in boreal forests.
The root and but rot caused by Heterobasidion annosum is one of the most destructive diseases of conifers in the northern hemisphere [2]. Despite biology and genetics of this fungus are well studied, the mechanisms of defense responses and resistance of pine trees are still unclear. Plant-pathogen interactions are well documented in crops, giving us the knowledge about gene regulation in those systems [3]. Evolutionary gymnosperm and angiosperm are quite distinct groups that separated from each other several hundred million years ago [4]. Despite their separation, main mechanisms and defense strategies towards pathogen invasion in both groups should be conserved [5]. Generally, in woody plants, plant defense mechanisms, comprise preformed and inducible physical and chemical barriers [5]. Preformed barriers are repre-
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sented by bark and cuticular waxes and chemical defenses comprise a range of antimicrobial compounds [6]. Recognition of potential pathogen triggers a cascade of biochemical reactions from primary and secondary metabolism [7]. Important place in defense response belongs also to the antimicrobial peptides which directly or indirectly involved in plant protection. Those proteins are widespread through the plant kingdom and are well studied in crops [8, 9]. Recently a lot of attention was attracted to role of those proteins in coniferous trees [10, 11, 12]. As few of them are well characterized in conifers, including Scots pine, functions of the rest remain uncertain. In this study we describe the development of the infection in pine saplings caused by H. annosum and evaluate the changes in transcription level of two groups of antimicrobial peptides: Scots pine lipid transfer protein (PsLTP1) and defensins (PsDef1-PsDef4).
Materials and methods. Sapling inoculation. Experiments were carried out from August 2011 to August 2012 using 3 years old Scots pine seedlings, grown on research field of UNFU Botanical garden. The H. annosum strain 1-10 was inoculated into the stem at 8 cm above the ground by cutting a 5mm small bark flap using a scalpel. A small amount of potato-dextrose agar (5x5x2 mm3) containing actively growing mycelium was placed underneath the bark flap, pressed firmly against the stem, and sealed with parafilm. The control was made in the same way but without mycelia. Both, control and experimental groups contained 10 seedlings. The infection was monitored in a few time periods: 1 month, half a year and one year after inoculation. After the experiment was over, the samples were collected and the cuts were made from inoculation zone, 2 cm above and 2 cm below the place of contact, to evaluate the development of the infection using light microscopy. The samples were examined at binocular and later the pictures were taken.
semiQRT-PCR. To measure the expression patterns of LTP and defensins, the samples were taken from infected and healthy saplings, as well as from the roots of 80 years old Scots pine trees, healthy and naturally infected. The levels of gene expression were checked on the mRNA level using a PCR technique. mRNA was obtained using modified method of lithium-chloride precipitation by Chang [13]. Primer pairs were picked up according to the sequences recently deposited by us in GenBank: PsLTP1 (Acc.No. JN980402.1), PsDef1 (Acc.No. EF455616.1), PsDef2 (Acc.No. EF455617.1), PsDef3 (Acc.No. JN980401.1) and PsDef4 (Acc.No. KJ601732.1). As a control for the reaction, we chose the house-keeping gene RPL44 (Acc.No. EL342388.1), which also was used for the calculation of the relative values of the expression level. PCR was run for 35 cycles in a thermal cycler using the program: 95OC, 1 min; 54OC,1 min; 72OC,1 min. The PCR products were electrophoresi-zed on a 1.5 % agarose gel and visualized with ethidium bromide staining and photographed. Densitometric analysis was run with Software GelProAnalyzed 4.0.
Results. Anatomical response of Scots pine stem tissues to fungus infected inoculation. Development of the pathological process causing inoculation with H. annosum at different time points was monitored by cross-sections (Fig. 1). One month after inoculation (August-September) was observed formation of cone-like necrotic zones (brown) traced to the pith. The inoculation site was abundantly soaked with resin.
HayKOBHH BicHHK HHTY YKpa'mn. - 2015. - Bun. 25.8
Fig 1. Development ofpathological process in the pine stems, infected with H. annosum mycelia at different periods. Cross sections of the pine stems infected at different time points and non-infected saplings n) necrotic zone; p) resin pocket; r) resin. Cut thickness 11.5 mm (Magnification 32 x. Division 1 = 0.5 mm)
The way of mycelia spreading, depends on the season, in which the pathological process takes place. During inoculation started on April till September, necrotic zone was localized on the pith with cone-like structure and primary sapwood has grown on the place of contact. Due to intensive cambium cells proliferation, the new wood layer with increased number of resin ducts was formed. In turn, this led to pitch-resin pocket formation, filled with resin inhibiting the spreading of mycelia into primary wood of current year. On pine seedlings crosscuts, where infection was developed at dormancy period, (end of September - March) extensive resin production was observed on damage areas as well as formation of a cone-like necrotic zone from bark to pith. In case, when infection was developed during a year starting from August, necrotic zone reached the pith stem and spread in wood of previous year. On the edge of annual rings the resin pocket was formed and dark pigmented spots appeared in the wood. Due to intensive proliferation of the cambium, the layer of sapwood with increased quantity of the resinoids filled with resin was formed, restricting the spreading of the mycelium onto the wood of the current year. In the sapwood of control plants, which were only wounded, formation of resinoids wasn't detected.
Analysis of the pathological process development in various seedlings showed variability in the resistance degree towards pathogenic fungi. On the Fig 2 is illustrated that the less resistant sapling (A1-A3) didn't have well-defined necrotic cone, its tissue accumulated a large amount of the resin and formed brown spots at some distance from inoculation point. On the contrary, the more resistant sapling (B1-B3) formed a clear conical area bounded by the core rays. No pigmented areas were observed at a distance of 2 cm.
AMP gene expression. During infection process was investigated the expression pattern of two groups of antimicrobial peptides, which supposedly are involved with plant defense reactions - lipid transfer protein and defensins, the last one included 4 genes (PsDefl, PsDef2, PsDef3 and PsDef4). To measure the changes in the expression pattern of defensins in three years old pine saplings infected with anno-
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sum root rot, the total RNA was extracted from the radial cuts of control and experimental samples, 5 of each. Infection in the stem has evolved over a month. Results are presented on a Fig.3.
Fig 2. Spreading of the root rot mycelium in the stem of 3 years old pine sapling one month post inoculation. A and B - independent samples. A1, B1 - stem cross-cuts made 2 cm above the inoculation zone; A2, B2 - stem cross-cuts from the inoculation zone; A3, B3 - stem cross-cuts made 2 cm below the inoculation zone (n - necrotic zone, r - resin)
Fig.3. Analysis of PsLTP1 and defensins expression in the Scots pine tissues of saplings (14) and adult trees (5, 6). A) electrophoregram of the RT-PCR products obtainedfrom RNA of 3 years old pine saplings inoculated with mycelia of H. annosum (2) from the zone of inoculation and 4- 2 cm above), and the control saplings inoculated without mycelium (1) from the zone of inoculation and 3-2 cm above); 5-6) expression patterns of samples obtainedfrom roots of 80 years old Scots pine healthy trees (5) and naturally infected with root sponge (6). M) GeneRuler 100 bp Plus DNA Ladder (Fermentas). Right arrows indicate the PCR-products: PsLTP1, PsDef1, PsDef2, PsDef3 and "house-keeping" gene RPL44. B) the values of the expression level of PsLTP1 calculated relative to RPL44.
HayKOBMM BicnnK II. IT Y YKpaiHM. - 2015. - Ban. 25.8
On the figure 3 is pointed out that expression levels of all investigated genes in control samples from the zone of inoculation and above the damage area show different expression patterns. In inoculation site was observed decreasing of the expression level of PsLTPl by 63 % compared to the corresponding control sample. The similar pattern was followed by defensins: the levels of PsDefl, PsDef2 and PsDef3 transcripts in the infected stems were significantly lower than in control sample. When measurements were taken above the inoculation area, the expression levels of PsLTPl, PsDefl, PsDef3 were 2.3-, 2.9-, 2.7-times lower respectively, while compared with control. PsDef2 transcripts level was on 70 % lower in sapwood of infected saplings than in corresponding control sample. Totally opposite results was obtained while checking the expression levels of PsLTPl and PsDef in roots of 80 years old pine. Three of four genes showed strong overexpression: levels of PsLTPl, PsDefl and PsDef3 transcripts were 2.2, 1.8 and 3.5 times higher in infected organs in comparison with non infected. As for PsDef2, the expression level in the root superficial parts of infected trees was lower by 75 % comparing with control. Besides defensin genes mentioned above, semi QRT-PCR was run also for another gene PsDef4, for which the same set of experiments was performed but no transcripts of this gene were present in examined plants.
Discussion. In wood trees the first line of defense towards different pathogens is presented by bark. Mechanical barrier of the strong lignified walls together with chemical properties of the phenolic compounds create multifunctional blockage for pathogens. But even tiny lesions on the bark can serve as a doorway for infection, especially if the last one is caused by aggressive decaying fungi such as root sponge, which appears to be very invasive wood destroyer. As we know, the spreading rate of the infection depends on many factors: stand type and history, forest composition, soil properties, including pH and the temperature [l6]. As known the infection process in pine caused by annosum root rot is well documented we decide to use that knowledge in our study. Using pure mycelia of the pathogen and applying it under the bark, we monitored the development of the pathologic process in young pine saplings. Previously Skipars used the method of the inoculation under the bark to estimate the resistance of individual adult trees to the annosum root rot. Mean length of the pigmented areas, above and below the inoculation zone, which developed in the phloem 3040 days after inoculation, served as a criterion for estimation of plant resistance [l7]. Our investigation show, that to measure resistance rate of the individual stands, it is necessary to estimate the spreading of the sponge rot not only on the vertical but also on the horizontal planes, as different tissues have different resistance rates.
Since infected samples were grown at the same edaphic conditions, equal in age and by morphometric criteria, and infected in the same way, obtained results show different resistance rates in each individual case towards annosum root rot. This can be explained by the differences in their genotypes, either it depends on physiology of each individual plant. Defense mechanism is very complex, so for it proper functioning well coordinated work of each link is a necessity.
At artificial infection, all protective mechanical barriers were destroyed, so the rest of the defense were passed to next level of the protection, in particular to the antimicrobial compounds involved in secondary metabolism, which are synthesized
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due to the activation of the specific genes expression [18]. Using transcriptomic analysis was shown that plant infection is followed with both over- and down regulation of a variety of genes involved into many key processes in plant physiology. Up-regulation revealed for genes involved into oxidative processes and in secondary metabolism, including phenylpropanoid pathway and down regulated genes mainly were linked to photosynthesis [19]. Dealing with the infection includes positive as well as negative regulatory mechanisms. Supposedly gene repression is associated with down regulation of non essential activities in cell and mobilization of all resources to deal with the challenge [7].
Defensins and LTP, examined in this study belong to different families of antimicrobial peptides: PR-12 and PR-14 respectively [20, 21]. There are a lot of data available about their role in wide range of cellular activities, including cell signaling, growth, development and plant defense [22, 23]. Previously we revealed that both de-fensin and LTP extracted from Scots pine seedlings show strong antifungal activity [11]. Thus evaluation of the expression pattern of those genes during advanced stages of annosum rot infection was very interesting by mean of finding correlation between genes expression and their possible role in defense. Annosum root rot inhibits expression of the PsDef1, PsDef2, PsDef3 and PsLTP in the sapwood of infected saplings in the place of inoculation as well as 2 cm above the inoculation area, which indicates that mentioned above genes aren't key elements in sapwood defense towards infection. Compared to young pines, in intact roots of adult tree infected with annosum rot the expression of PsDef1, PsDef3 and PsLTP is very high in contrast to healthy plant. Probably the natural infection in adult pine is hitting the effective defense barriers, but as we don't know the period when infection occurred and how long it was developing, it is hard to say if such high level of AMP transcripts is a result of strong immunity reaction or either the reason lies in the tree genotype.
References
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11. Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings / V. Kovaleva, R. Kiyamova, R. Cramer та ш]. // Peptides. - 2009. - № 30. - Pp. 2136-2143.
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13. Chang S. A Simple and Efficient Method for Isolating Rna from Pine Trees / S. Chang, J. Pur-year, J. Cairney. // Plant Molecular Biology Reporter. - 1993. - Vol. 11, № 2. - Pp. 113-116.
14. Hrunyk N. Patterns of expression of lipid-transfer protein gene in organs of Scots pine (Pinus sylvestris L.) / N. Hrunyk, V. Kovaleva, R. Gout. // Studia Biologica. - 2012. - Vol. 6, № 2. - Pp. 151-160.
15. The effect of phytohormones on expression of defensin gene in Scots pine / Y. Shalovylo, Y. Yusypovych, V. Kovaleva, R. Gout. // Studia Biologica. - 2015. - Vol. 9, № 1. - Pp. 15-24.
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ГруникН.1., Юсипович Ю.М., Ковальова В.А., Гут Р.Т. Розвиток ко-ренево'1 гнил та ощнювання р1вм1в експреси лшвдтрансферного протешу та дефензишв у сосни звичайно! шд впливом Heterobasidion annosum
Збудник коренево! та комлево! гнилизни - Heterobasidion annosum - е одним 1з найнебезпечнших патогешв хвойних порщ, який спричиняе масове всихання бореаль-них л1С1в у пшшчнш швкул1. Незважаючи на те, що бюлогвд та генетику цього гриба вивчено достатньо добре, мехашзми захисту та стшкост соснових дерев залишаються нез'ясованими. Останш досягнення у вивченш транскриптому системи коренева губка-сосна та молекулярной характеристик фактор1в патогенност наблизили нас до розумш-ня ще! патосистеми. Дослщжено особливосй розвитку патолопчного процесу в с}янцях сосни звичайно!, шфшованих кореневою губкою у р1зш перюди !хнього росту, а також, за допомогою нашвкшьюсно! ЗТ-ПЛР, встановлено профш експреси лшщтрансферно-го протешу та дефензинш у здорових та шфшованих рослин.
Ключовi слова: сосна звичайна, дефензин, лшщтрансферний проте!н, експресш, коренева гнилизна.
Грунык Н.И., Юсипович Ю.М., Ковалева В.А., Гут Р.Т. Развитие корневой гнили и оценка уровней экспрессии липидтрансферного протеина и дефензинов у сосны обыкновенной под влиянием Heterobasidion annosum
Возбудитель корневой и комлевой гнилей - Heterobasidion annosum - является одним из наиболее опасных патогенов хвойных пород, который вызывает массовое усы-хание бореальных лесов в северном полушарии. Несмотря на то, что биология и генетика этого гриба изучены достаточно хорошо, механизмы защиты и стойкости сосновых деревьев остаются невыясненными. Последние достижения в изучении транскриптома системы корневая губка-сосна и молекулярной характеристике факторов патогенности приблизили нас к пониманию этой патосистемы. Изучены особенности развития пато-
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Нащональний лкотехшчний унiверситет Украши
логического процесса в сеянцах сосны обыкновенной, инфицированных корневой губкой в разные периоды их роста, а также установлены профили экспрессии липидпере-носящего белка и дефензинов у здоровых и инфицированных растений с помощью полуколичественной ОТ-ПЦР
Ключевые слова: сосна обыкновенная, дефензин, липидпереносящий белок, экспрессия, корневая гниль.
УДК 630.284.2 Доц. Л. С. Осадчук, д-р с.-г. наук - НЛТУ Украти, м. Львiв
ВИКОРИСТАННЯ МЕТОДУ М1КРОПОРАНЕННЯ ДЛЯ ПРОГНОЗУВАННЯ СМОЛОПРОДУКТИВНОСТ1 ДЕРЕВ СОСНИ ЗВИЧАЙНО1
Виявлено прямолшшний достовiрний сильно! псноти зв'язок мiж виходом живищ з мiкропоранення та з карошдновки. Визначення смолопродуктивностi методом мшро-поранення дае змогу встановити категорiю смолопродуктивност дерев, а через ршнян-ня залежностi оцiнювати смолопродуктивнiсть окремих дерев та !х сукупностi з достат-нiм ступенем точностi. Практично вс дерева, що належать до певно! категори смолоп-родуктивностi за виходом живищ, потрапляють в ту ж категорвд i за мiкропораненням. Помилки в розподiлi виявлено у 8 % дерев низько!, 68 % середньо! та 5 % високо! категорий смолопродуктивностi, якi за виходом живищ переважно знаходяться на межi су-сiдньо'í категори.
Ключовi слова: метод мшропоранення, смолопродуктивнiсть, сосна звичайна.
Вступ. Свiтове виробництво канiфолi та скипидару та ринки !х збуту на сьогоднi пов'язанi з Китаем, який е найбшьшим у свт виробником, спожива-чем й експортером продукпв пiдсочного виробництва - це приблизно 70 % вщ свiтового виробництва. На сьогоднi зростае попит у свт i в бврош на еколопч-но чисп продукти з живицi, зокрема, у виробнищш бiопалива, у медицинi, у харчовш промисловостi та парфумерií, що веде до стимуляцп виробництва у цш галузi загалом [8-11]. Позитивним фактором е також новi розробки з мехаш-зацií та системи заготiвлi живицi з метою тдтримки i розширення цього виробництва [4, 12, 13].
Потенцшш можливосп пiдсочки хвойних насаджень Украши, передуам у соснових лiсах, е значними. Одним iз можливих i економiчно виправданих напрямкiв подальшого удосконалення заготiвлi сосново! живицi, понижения ц собшартосп та росту продуктивностi працi шд час ц заготiвлi е вiдбiр високос-молопродуктивних дерев [3, 7].
Матерiали i методика дослiджень. Для досягнення достатньо! точностi дослiду проведено поршняння смолопродуктивностi у 100 дерев. На однш сто-ронi стовбура смолопродуктивнiсть визначали за звичайно! шдсочки з карошд-новки за кшьктстю живицi у приймачi [5]. Смолопродуктивнкть на одну каро-пiдновку визначено за 10 обходiв з паузою 3,5 дшв i кроком пiдновки 12 мм. Вихщ живицi вираховували шдивщуально в кожного дерева з точнктю до 1 г. Пiзнiше розраховували коефiцiеити смолопродуктивностi для кожного дерева. На шшш сторонi стовбура для визначення смолопродуктивносп застосо-вували метод мiкропоранения: на висотi 1,3 м спещальним свердлом наносили поранення глибиною i дiаметром у деревиш 5 мм. Вихвд живицi методом мжро-поранення встановлювали за добу, вимрюючи довжину трубки, заповнено! жи-
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Збiрник науково-техшчних праць
