ALEKSANDRAS STULGINSKIS UNIVERSITY

LITHUANIAN RESEARCH CENTRE FOR AGRICULTURE AND FORESTRY

Rūta Česnulevičienė

HARMFULNESS OF FIELD PEA (Pisum sativum L.) FUNGAL DISEASES, THEIR PREVENTION AND CONTROL

Summary of Doctoral Dissertation Agricultural Sciences, Agronomy (01A)

Akademija, 2012 The research was carried out at the Lithuanian Research Centre for Agriculture and Forestry in 2008–2011

Scientific supervisor: Irena Gaurilčikienė (Lithuanian Research Centre for Agriculture and Forestry, agricultural sciences, agronomy – 01A)

The doctoral dissertation will be defended at Aleksandras Stulginskis University Agricultural Sciences Council:

Chairman: Prof. habil. Dr. Zenonas Dabkevičius (Aleksandras Stulginskis University, agricultural sciences, agronomy – 01A) Members: Dr. Irena Brazauskienė (Lithuanian Research Centre for Agriculture and Forestry, agricultural sciences, agronomy – 01A) Prof. habil. dr. Romualdas Juknys (Vytautas Magnus Universitety, biomedical sciences, ecology and environment – 03B) Habil dr. Juozas Staniulis (Nature Research Centre, biomedical sciences, biology – 01B) Prof. habil dr. Gediminas Staugaitis (Lithuanian Research Centre for Agriculture and Forestry, agricultural sciences, agronomy – 01A) Oponents: Assoc. prof. dr. Ingrida Šiaulienė (Šiauliai University, biomedical sciences, ecology and environment – 03B) Dr. Alma Valiuškaitė (Lithuanian Research Centre for Agriculture and Forestry, agricultural sciences, agronomy – 01A)

The doctoral dissertation will be defended at the public session held by Council of Agricultural Sciences at 11 a.m. on June 25, 2012 at Aleksandras Stulginskis University in room 261. Adress: Studentų str. 11, LT-5361 Akademija, Kaunas district, Lithuania.

The abstract of the doctoral dissertation was distributed on May 25, 2012. The dissertation is available at the libraries of Aleksandras Stulginskis University and Lithuanian Research Centre for Agriculture and Forestry.

2 ALEKSANDRO STULGINSKIO UNIVERSITETAS LIETUVOS AGRARINIŲ IR MIŠKŲ MOKSLŲ CENTRAS

Rūta Česnulevičienė

SĖJAMOJO ŽIRNIO (Pisum sativum L.) GRYBINIŲ LIGŲ ŽALINGUMAS, JŲ PREVENCIJA IR KONTROLĖ

Daktaro disertacijos santrauka Žemės ūkio mokslai, agronomija (01A)

Akademija, 2012

3 Disertacija rengta 2008–2011 m. Lietuvos agrarinių ir miškų mokslo centre.

Mokslinis vadovas: Dr. Irena Gaurilčikienė (Lietuvos agrarinių ir miškų mokslo centras, žemės ūkio mokslai, agronomija – 01A)

Disertacija ginama Aleksandro Stulginskio universiteto Agronomijos mokslo krypties taryboje:

Pirmininkas:

Prof. habil. dr. Zenonas Dabkevičius (Aleksandro Stulginskio universitetas, žemės ūkio mokslai, agronomija – 01A) Nariai: Dr. Irena Brazauskienė (Lietuvos agrarinių ir miškų mokslo centras, žemės ūkio mokslai, agronomija – 01A) Prof. habil. dr. Romualdas Juknys (Vytauto Didžiojo universitetas, biomedicinos mokslai, ekologija ir aplinkotyra – 03B) Habil dr. Juozas Staniulis (Gamtos mokslų centras, biomedicinos mokslai, biologija – 01B) Prof. habil. dr. Gediminas Staugaitis (Lietuvos agrarinių ir miškų mokslo centras, žemės ūkio mokslai, agronomija – 01A) Oponentai: Doc. dr. Ingrida Šiaulienė (Šiaulių universitetas, biomedicinos mokslai, ekologija ir aplinkotyra – 03B) Dr. Alma Valiuškaitė (Lietuvos agrarinių ir miškų mokslo centras, žemės ūkio mokslai, agronomija – 01A)

Disertacija bus ginama viešame Agronomijos mokslo krypties tarybos posėdyje 2012 m. birželio 25 d. 11 val. Aleksandro Stulginskio universiteto 261 auditorijoje.

Adresas: Studentų 11, LT-53361 Akademija, Kauno raj., Lietuva. Disertacijos santrauka išplatinta 2012 gegužės 25 d.

Disertaciją galima peržiūrėti Aleksandro Stulginskio universiteto ir Lietuvos agrarinių ir miškų mokslo centro bibliotekose.

4 INTRODUCTION Relevance of the subject. The field pea (Pisum sativum L.) is valued for its protein-rich grain and for being a good pre-crop for other crops. In Lithuania, over the last decade, the field pea production area has fluctuated from 7.4 thousand ha in 2003 to 27.0 thousand ha in 2011. The crop productivity varied significantly, too from 1.07 t ha-1 in 2006 to 2.0 t ha-1 in 2009 (http://faostat.fao.org). One of the reasons responsible for the low pea productivity is fungal diseases causing significant annual losses in grain yield (Garry et al., 1998, Beasse et al., 2000, Xue, 2000). More than 20 fungi species infesting pea crops have been established in various parts of the world. Ascochyta blight is widespread in all pea growing regions. Its causal agents are Ascochyta pisi, Mycosphaerella pinodes, Phoma pinodella, which are often referred to in literature as Ascochyta complex (Onfroy et al., 1999). These pathogens damage plant root, foot and aerial part, pods and grains (Walen, 1965; Maude, 1966; Moussart et al., 1998; Bretag et al., 2006; Marcinkowska, 2008). Apart from the diseases caused by the pathogens of the Ascochyta complex, an array of other fungi, which occur singly or in various combinations, parasitize pea. Aphanomyces euteiches, which causes root rot, is widespread in France (Wicker et al., 2001), the Netherlands (Oyarzun, van Loon, 1989), Southern Scandinavia (Persson et al., 1997) and many other pea growing regions around the world. In Denmark and Sweden, the most frequently isolated pathogens from pea roots were P. pinodella and Fusarium solani, other pathogens involved in root rot complex were F. avenaceum, F. oxysporum, F. culmorum, Chalara elegans, Pythium irregulare and M. pinodes (Persson et al., 1997), in Canada – Fusarium spp. and Pythium fungi (Hwang, Chang, 1989). In Lithuania, pea pathogens Ascochyta pinodes, A. pisi, Botrytis cinerea, Colletotrichum pisi, Didymella pinodes, Erysiphe pisi, Fusarium spp. have been first mentioned in the Conspectus of the Parasitic Mycoflora of Leguminous Plants in the Baltic Region (Струкчинскас, 1980). The findings of research conducted in 2006 and 2007 suggested P. pinodella and Fusarium spp. to be the prevalent causal agents of pea root rots, less frequent were Botrytis spp., Thielaviopsis basicola, Rizoctonia spp., and the ones identified on the aerial plant part were A. pisi, M. pinodes, P. pinodella, Erysiphe pisi var. pisi, Peronospora viciae f.sp. pisi (Gaurilčikienė, Janušauskaitė, 2007). A reduction in the content of A. euteiches, Pythium spp. and other soil pathogen inoculum can only be achieved through the application of appropriate crop rotation; however, even after a 6-8 year break, severe outbreaks of root rots are plausible (Pfender, Hagedorn, 1983). The causal agents of ascochyta blight A. pisi, M. pinodes, P. pinodella are transmitted through infected seed and plant residues in the soil. The conidia of all pathogens of Ascochyta complex are dispersed by rain-splash in a crop within a short distance; however, M. pinodes ascopores are also wind-dispersed. The disease progression in a cop is significantly influenced by the plant architecture of the pea varieties and plant morphological peculiarities: leaf area and distribution, internode length, branching, stem strength and height (Schoeny et al., 2008; Le May et al., 2008). In Canada, where the pea cultivation area totals 1.3 million hectares, the yield losses from the diseases caused by Ascochyta complex amount to 50% and in separate years to 70% (Wallen, 1974; Xue, 2003). In France, research evidence has shown that in 7

5 years out of 10 pea grain yield losses from ascochyta blight (M. pinodes) amounted to 20% (Beasse et al., 2000). The abundance of pathogens causing pea root rots makes breeding for root rot resistance complicated. No pea varieties resistant to the soil pathogens A. euteiches, Fusarium spp. and others have been developed yet. Different pea pathogens differ in resistance to ascochyta blight; however, none of the existing varieties has exhibited complete resistance. Quantitative trait loci (QTL), influencing ascochyta blight resistance have been identified in a pea genome but molecular mechanism of resistance still has not been well ascertained (Onfroy et al., 1999; Wang et al., 2000; Timmerman- Vaughan et al., 2002; Prioul-Gervais et al., 2007). The susceptibility of Lithuania- grown pea varieties to root and foot rots and ascochyta blight is not known; moreover, there are no scientifically validated recommendations concerning measures and practices designed for the reduction of damage done by fungal diseases in pea crops. Research hypothesis. The incidence and harmfulness of root and foot rots and ascochyta blight depend on pea genotype and agro-ecological factors. Seed treatment can reduce the severity of root and foot rots. It is plausible that fungicide spray- application at pea flowering stage can reduce ascochyta blight harmfulness and enhance pea productivity. Research objective and experimental tasks. The study was designed to explore the incidence and severity of root and foot rots and ascochyta blight in field pea crops and to identify the measures and practices for the prevention and control of the diseases caused by the pathogens of Ascochyta complex. Experimental tasks: - To identify the susceptibility of various field pea varieties to root and foot rots and ascochyta blight under different agro-ecological conditions. - To establish the effect of meteorological factors on the severity of root and foot rots and ascochyta blight in field pea crops. - To determine the frequency of detection of pathogens of Ascochyta complex on various pea varieties. - To estimate the feasibility of control of the diseases caused by the pathogens of Ascochyta complex using seed treatment and fungicide application. - To assess the impact of seed treatment and fungicide application on field pea productivity and yield components. - To study the possible side-effect of the chemical seed treatment on the microflora of pea rhizosphere and soil. Originality of the research work. This study is the first attempt in Lithuanian to ascertain the development of field pea root and foot rots and ascochyta blight severity in relation to the genotype and meteorological factors. Detection frequency of the pathogens of Ascochyta complex, which has not been investigated before, was established in semi-leafless pea at different ontogenesis stages of plants. Efficacy of the control of the diseases caused by these pathogens was explored using seed treatments and fungicidal spray applications on crops, and the influence of the tested measures and practices on pea productivity and yield components was estimated. The seed treatment agents were found to exert a negative side-effect on the micro-fungi and a positive effect on bacteria of pea rhizosphere and soil. 6 Practical relevance of the work. The current research revealed the field pea varieties, less susceptible to root and foot rots and ascochyta blight, which could be grown under sustainable and organic cropping systems. The tested control measures of the diseases caused by Ascochyta complex: seed treatment reduced the severity of root and foot rots until the flowering stage; fungicidal spray applications on pea crops inhibited the incidence of ascochyta blight on pods and grain. The practical implementation of the preventive and control measures of Ascochyta complex will enable reduction of pea grain yield losses. Propositions to be defended: - The susceptibility of the field pea varieties to root and foot rots and ascochyta blight depends on the genotype and agro-ecological conditions. - Meteorological conditions exert a marked effect on the severity of root and foot rots and ascochyta blight in field pea crops. - The pathogens of Ascochyta complex are identified on pea plants since seedling stage and during the growing season infect the whole plant. - Reduction of harm done by the diseases caused by the pathogens of Ascochyta complex is feasible through the protection of seedlings from seed-borne infection and through fungicidal spray applications on crops. - Seed treatment and fungicidal spray applications on crops increase pea productivity. - The use of chemical agents for seed treatment can have a negative side-effect on the micro-organisms of pea rhizosphere and soil. Approval of the experimental results. The major research results were published in 2 scientific articles, in publications indexed in the ISI WOS data base, 4 articles published in conference proceedings and one recommendation was prepared for agricultural producers. The research findings were presented in the international conferences “Soil stability in ecologically and socially vulnerable regions” (Perloja, 2009, oral presentation), “Advances in plant protection strategies” (Druskininkai, 2008, poster presentation), international symposium “Seed Health in Agricultural Development” (Beijing, China, 2010, poster presentation), conference “Agricultural and forestry sciences: recent research results and innovative solutions” (Akademija, 2011, oral presentation). Volume of the dissertation. The dissertation is written in the Lithuanian language, its volume is 101 pages, including 36 tables and 18 figures. A total of 246 literature references have been used. EXPERIMENTAL CONDITIONS AND METHODS Determination of field pea varieties’ susceptibility to root and foot rots and ascochyta blight under different agro-ecological conditions. During the period 2008–2010, in different soil and climate conditions: 1) in Middle Lowland of Lithuania in Dotnuva, 2) in Southeast Lithuania in Perloja, experiments were conducted to investigate the development of severity of root and foot rots and ascochyta blight in the crops of semi-leafless field pea varieties Profi, Eiffel, Simona, Tinker, Mascara and Pinochio. In the crops of the same varieties, research was done into the detection 7 frequency of the pathogens of Ascochyta complex on plants at different stages of their ontogenesis. In both experimental sites (Dotnuva and Perloja), experiments were sown using the same untreated seed at a rate of 1 million viable seeds ha-1, a conventional technology for pea cultivation for grain was employed. For all varieties tested, 6x24 m observation plots were formed in 6 metre-wide bands, where from plant emergence to maturity, assessments of root and foot rots and ascochyta blight severity were carried out periodically, every 14-18 days. For disease pressure assessment, 3 samples of 15 plants were formed from each variety’s observation plot, phenological development stage was determined according to BBCH scale (Weber, Bleiholder, 2001). In 2008, the severity of root and foot rots was estimated according to 0-3 point scale (Cooke, 2006), in 2009-2010 according to 0-5 point scale (Garry et al., 1996; Grünwald et al., 2003). The severity index (%) of root and foot rots was calculated after Gilpatrick, Bush (1950). Ascochyta blight severity on plants and pods was estimated according to the disease-affected plant surface according to percentage scale (Slopek, 1989; Sharma, 2004). The severity of root and foot rots and ascochyta blight per season is expressed by the AUDPC (area under disease progress curve) value (Campbell, Madden, 1990). Frequency of detection of pathogens of Ascochyta complex was investigated by cultivating fragments of plant parts or grains on an oat agar medium (Dhingra, Sinclair, 1994; Roger, Tivoli, 1995). The pathogens of Ascochyta complex were identified according to the morphological traits typical of the colonies (Punithalingam, Holliday, 1972a; Punithalingam, Holliday, 1972b; Punithalingam, Gibson, 1976; Mathur, Kongsdal, 2003). Frequency of detection of Ascochyta complex pathogens and A. pisi relative density in Ascochyta complex were calculated (González ir kt., 1995). Control of the diseases, caused by the pathogens of Ascochyta complex, using seed treatment and fungicide application. During 2008–2010, precision field trials were set up in Dotnuva and Perloja. The experimental design involved seed treatment and fungicide application in crops. In all experimental years, the seed of the pea variety Pinochio was sown. Commercial seed from the same lot was sown in Dotnuva and Perloja. Two seed treatment agents were chosen for the study – Raxil extra (a. i. tebuconazole+thiram 15+500 g l-1) and Kinto (a.i. triticonazole+prochloraz 20+60 g l-1) and 2 fungicides – Signum (a. i. boscalid+piraclostrobin 267+67 g kg-1) and Bravo (a.i. chlorothalonil 500 g l-1). The rates of seed treatment agents and fungicides are provided in Table l. Table 1. Experimental design of field trials. Dotnuva, Perloja, 2008-2010. Rate of seed Rate of fungicide Treatment Seed treatment/fungicide treatment l, kg ha-1 No. ml 100 kg-1 seed 1. Untreated / Without fungicides - - 2. Untreated / Signum - 1.0 3. Untreated / Bravo - 3.0 4. Kinto / Without fungicides 150 - 5. Raxil extra / Without fungicides 200 - 6. Kinto / Signum 150 1.0 7. Raxil extra / Signum 200 1.0 8. Kinto / Bravo 150 3.0 9. Raxil extra / Bravo 200 3.0 8 Pea seed was treated with a seed treating machine Amazone (Germany), with the water slurry of 1 l 100 kg-1. The experimental plots (3x20 m) were sown with a drilling machine Fiona (Denmark) with a row spacing of 12.5 cm, 4 replications, at a seed rate of 1 million viable seed ha-1. Fungicides were sprayed at pea flowering stage (BBCH 61-65) with a precision, small-plot bicycle sprayer using compressed nitrogen, flat-fan nozzles 4110-12. The assessments of the severity of pea root and foot rots were started at seedling stage and those of ascochyta blight at the time of disease appearance in the crop and were continued every 14-18 days until maturity. The severity was estimated in the same way as of the above described plants of the pea varieties and the data were analysed also in the same way by calculating severity index (%) of rots, ascochyta blight severity (%) and AUDPC values. The method of wet blotter paper rolls was used for studying seed-borne pea seedling infection (ISTA. International rules for seed testing, 1996). In this test we used Pinochio seed –untreated and treated with Raxil extra and Kinto. The plants were incubated for 21 days at 12 h light photoperiod at 20°C temperature. The blackened or browned seeds, seedlings with brown, black spots or stripes on hypocotyl, epicotyl or rootlets were considered to be affected by pea seed infection. The effect of seed treatment and fungicides on field pea productivity and yield components. At pea grain maturity stage, before harvesting, sheaves of 50 (5x10) pea plants were formed from each plot for the determination of pod number per plant, grain number and weight per plant (g). Grain yield was harvested at complete maturity with a combine harvester Wintersteiger Delta (Germany) in Dotnuva and with Sampo 500 (Finland) in Perloja. The harvested grains were weighed and 2 kg samples were taken for the establishment of grain moisture content and 1000 grain weight (TGW), and seed infection with the pathogens of Ascochyta complex. Side-effects of chemical seed treatment on the micro-flora of pea rhizosphere and soil were investigated in the field trial in Dotnuva. Soil samples from the plots sown with untreated seed (treatment 1), Kinto-treated seed (treatment 4) and Raxil extra-treated seed (treatment 5) were taken at pea seedling growth stage BBCH 11-12 and bud formation stage BBCH 51-55 between plant rows (at the 0-7.5 cm depth) and were referred to as bulk soil, and from the soil in plant rows referred to as the soil from rhizosphere. For soil bacteria and fungi quantification we used dilution method and selective media. The number of bacteria was determined after 4 days and that of fungi after 7 days. The final number of colony forming units (cfu) was adjusted according to soil moisture by recalculating into cfu g-1 dry soil. Statistical data analysis. The experimental data were processed by the analysis of variance method. The significant difference of data was estimated according to Fisher and Duncan criteria. The method of binary regression and correlation analysis of data was used to estimate the interaction between AUDPC of root and foot rots and ascochyta blight and environmental factors and disease AUDPC and yield and its elements. Statistical analysis was done using the statistical data processing software package ‘Selekcija’ (software ANOVA, STAT ) (Tarakanovas, Raudonius, 2003). Meteorological conditions. In 2008, in Dotnuva the sum of effective temperatures (∑≥5°C) was significantly higher than that in Perloja nearly throughout the whole growing season; however, the amount of rainfall was markedly higher in Perloja. In 2009, a slightly higher sum of effective temperatures since early spring was recorded in 9 Dotnuva, while in summer months in Perloja. The year 2009 was distinguished by a droughty spring, especially in the Middle Lithuania zone. Later in the season, both experimental sites received a similar amount of rainfall, except for the downpour in Dotnuva, where 74 mm of rainfall fell within 4 hours on June 23. In 2010, unlike in the previous year, there was more rainfall and rainy days in spring in Dotnuva; however, the first two ten-day periods of June and July were wetter in Perloja, except for July the third ten-day period when Dotnuva received as much as 101 mm more rainfall than Perloja. The effect of the meteorological factors – amount of precipitation (mm) and sum of effective temperatures (∑≥5 °C) on the severity of root and foot rots and ascochyta blight in field pea was estimated by a binary regression and correlation data analysis method. RESULTS The susceptibility of field pea varieties to root and foot rots under different agroecological conditions. In 2008, root and foot rots appeared in the crops of pea varieties at seedling stage both in Perloja and Dotnuva. The severity of rots consistently increased until flowering stage, later progressed more rapidly and at maturity stage roots and foot were severely affected. The plants of Mascara variety were the most susceptible to root and foot rots both in Dotnuva and Perloja, while Tinker was severely affected in Perloja, but in Dotnuva this variety was one of the least affected ones. Pinochio plants were more tolerant of root and foot infection. In 2009, in Perloja, the severity index of root and foot rots increased from 25.3- 54.0 % at bud formation stage within 14 days to 68.0-90.7 % at flowering stage. Until bud formation stage, the least affected varieties were Pinochio and Eiffel, while the most severely affected ones, like in 2008, were found to be Tinker and Mascara. At grain forming-maturity stage, all plants were very severely affected by the rots. In Dotnuva, the severity of rots increased gradually and at maturity stage the roots and foot of all pea varieties were moderately affected (51.3-66.0 %), except for Mascara variety whose foot rot index was 81.4 %. In the spring of 2010, both in Perloja and Dotnuva sites, the conditions were very conducive to plant growth, and the spread of root and foot rots was slow until flowering stage. In Perloja, the roots and foot of Tinker and Mascara plants were slightly more intensively affected. The severity of rots markedly increased only at maturity stage. In Dotnuva, the severity of root rots progressed very rapidly from weak (21.3-34.7 %) at flowering stage to extremely severe (100 %) at the end of grain-forming stage. The severity of foot rots increased less rapidly, the severity index increased from 32.0- 48.0% at the beginning of flowering to 74.3-84.0 % during maturity stage. Mascara plants were most severely affected, while Profi and Simona were less susceptible. In 2009, the AUDPC values of root and foot rots in Perloja were the highest while they were the lowest there in 2010, when the incidence of rots in the first half of the growing season was low (Table 2). The results of the three experimental years from the two experimental sites indicated that in Perloja the highest AUDPC values of root rots were recorded for Tinker and Mascara, while in Dotnuva for Mascara. In two years out of three, the lowest AUDPC values, significantly differing from the most severely affected in Perloja Tinker and Mascara, were identified for Profi, Eiffel, Simona and Pinochio. In Dotnuva, the AUDPC values of root rots significantly lower than those for 10 Mascara, were identified for Pinochio in all three experimental years, and for Profi and Tinker in two experimental years. The severity of foot rots in the crop of Tinker variety was particularly dependent on the agro-ecological conditions of the experimental site. Table 2. The AUDPC values of root and foot rots of field pea in the two experimental sites – Perloja and Dotnuva Variety 2008 2009 2010 2008 2009 2010 AUDPC of root rot AUDPC of foot rot Perloja Profi 1933 ab 2653 a 1358 a 1680 a 2638 c 1306 b Eiffel 1977 ab 2556 a 1363 a 1712 a 2400 a 1024 a Simona 2027 b 2541 a 1297 a 1740 a 2453 abc 1015 a Tinker 2400 d 2677 a 1824 c 2286 c 2636 bc 1501 bcd Mascara 2405 d 2589 a 1814 c 2122 c 2566 abc 1651 d Pinochio 1728 a 2515 a 1377 a 1577 a 2536 abc 1349 b Dotnuva Profi 1829 a 1479 b 1933 a 1583 a 1566 c 1441 a Eiffel 2116 bc 1479 b 2004 a 1820 d 1422 bc 1643 ab Simona 2125 c 1378 ab 1974 a 1814 cd 1255 a 1599 ab Tinker 1826 a 1451 ab 1963 a 1583 a 1312 ab 1905 cd Mascara 2055 bc 1694 c 2208 b 1790 bcd 1930 d 1950 d Pinochio 1749 a 1339 a 1985 a 1632 ab 1359 ab 1713 bcd The values denoted by the same letter in the same site and year did not differ significantly (P≤0.05). In our study, especially in 2009, we noted a particularly marked effect of the site’s meteorological conditions on the severity of root and foot rots in different field pea varieties. Pea roots and foot are affected by a complex of pathogens. The prevalent pathogens are A. pinodes, M. pinodella and Fusarium spp. (Hwang et al., 1995; Persson et al., 1997; Moussart et al., 1998; Gaurilčikienė, Janušauskaitė, 2007). The pathogens of Ascochyta complex - A. pinodes, M. pinodella affect pea plants already at seedling stage, both of these fungi are characterised by an extremely high rate of transmission from seed into emerging plant, and they cause rots and later stem base blackening (Wallen ir kt., 1967a; Moussart et al., 1998; Peck et al., 2001; Marcinkowska et al., 2009). Development of ascochyta blight-resistant varieties has become breeders’ focus worldwide; however, so far we have been aware of the existence of the varieties that are tolerant of only pathogens of Ascochyta complex, and which are considered to be less affected by the pathogen-caused foot rots and prevent the spread of disease on aerial plant parts only in the case of low infection pressure (Kraft et al., 1998; Xue, Warketing, 2001; Gossen et al., 2011). The abundance of pathogens causing root rots makes breeding for rot resistance complicated (Grünwald et al., 2003; Infantino et al., 2006). The susceptibility of field pea varieties to ascochyta blight under different agroecological conditions. In 2008, in Perloja the first symptoms of ascochyta blight were spotted in the crops of Tinker and Mascara varieties at stem elongation stage (BBCH 33-35), the lowest ascochyta blight severity at maturity stage was recorded for Simona variety. In Dotnuva, ascochyta blight appeared much later, the first disease

11 symptoms on Profi and Pinochio plants were spotted only at flowering stage (BBCH 63), while on Simona the disease occurred as late as seed ripening stage (BBCH 85). In 2009, in Perloja, ascochyta blight started to spread in pea crops at the end of bud formation stage, and by the beginning of flowering (BBCH 61-62) the disease had already severely spread in the crops of all varieties. That year, significantly highest disease severity was identified for Mascara and Tinker, while the lowest severity was measured for Simona and Pinochio. In Dotnuva, in 2009, the severity of ascochyta blight was markedly lower than that in Perloja, and the disease manifested itself more strongly only at seed ripening stage (BBCH 83). The plants of Profi were most severely affected. In 2010, both in Perloja and Dotnuva, ascochyta blight started to spread early, the first disease symptoms in the crops of all varieties were spotted at stem elongation stage (BBCH 36-37). The disease severity until beginning of flowering (BBCH 61) was low; later, at grain-formation stage (BBCH 79), in Perloja ascochyta blight significantly more severely affected Tinker, while Simona plants were least affected. In Dotnuva also the highest ascochyta blight severity was identified on Tinker pea plants, the disease progressed more slowly in the crops of Pinochio, Mascara and Simona varieties. The incidence of ascochyta blight on the pods of various pea varieties was observed in all experimental years. In 2008, the pods of Simona and Pinochio varieties were free from ascochyta blight in both experimental sites. In 2009, lower disease pressure was noted on the pods of Pinochio (73.3 %) in Perloja, in Dotnuva on the pods of Tinker (66.7 %). In 2010, in Perloja ascochyta blight incidence on pods was as high as 95.0- 100%, in Dotnuva 75.0-95.0 %. The disease severity was particularly significantly influenced by the experimental site’s agro-ecological conditions. In all experimental years, the AUDPC values of ascochyta blight were higher in Perloja than those in Dotnuva (Table 3). The AUDPC values of the disease were the highest for Tinker in Perloja and for Profi in Dotnuva for two years out of three, while for Simona these values were the lowest. The AUDPC values of ascochyta blight for the other varieties tested varied markedly between years and experimental sites. The data of the experiments carried out for three years in different agro-climatic conditions showed Tinker to be more susceptible to ascochyta blight, while Simona and Pinochio varieties were less susceptible. Table 3. The AUDPC values of ascochyta blight of field pea in Dotnuva and Perloja Variety Perloja Dotnuva 2008 2009 2010 2008 2009 2010 Profi 99 ab 271 c 455 ab 37 c 132 c 102 c Eiffel 152 cd 257 bc 463 ab 27 abc 70 ab 92 bc Simona 83 a 253 bc 385 a 19 a 48 a 90 bc Tinker 169 d 260 bc 601 d 27 abc 78 ab 175 d Mascara 133 bcd 251 bc 414 ab 15 a 59 ab 72 ab Pinochio 100 ab 216 a 516 bcd 21 a 91 b 65 a The values denoted by the same letter in the same site and year did not differ significantly (P≤0.05).

The effect of the meteorological factors on the severity of root and foot rots and ascochyta blight in field pea crops. A combination of three factors is necessary for the spread of fungal diseases: pathogen – host plant – favourable environmental 12 conditions (Van der Plank, 1963). In our study, in both experimental sites (Dotnuva and Perloja) the first two factors were similar; however, there were annual differences in the meteorological conditions (air temperature, precipitation rate as well as the number of rainy days) between the sites (Dotnuva - Middle and Perloja - Southeast Lithuania). Because of the differences in the environmental parameters between the sites the incidence of pea root and foot rots and ascochyta blight was uneven. To estimate the effects of the amount of precipitation and the sum of effective temperatures (∑≥5 °C) on the AUDPC values of root and foot rots and ascochyta blight, a binary regression correlation analysis of data was conducted. In Dotnuva, the AUDPC values of root rots of the tested pea varieties were found to be particularly strongly positively correlated with the amount of rainfall in the second half of the growing season, while in Perloja with the amount of rainfall in the first half of the growing season (Table 4). In both experimental sites, the AUDPC values of root rots strongly correlated with the sum of effective temperatures for Tinker and Mascara, while the AUDPC values of foot rot strongly correlated with sum of effective temperatures for Eiffel, Simona and Tinker. A strong significant correlation (P≤0.01) was established among the AUDPC values of pea ascochyta blight and the sum of effective temperatures during the growing season and the amount of precipitation in the second half of the growing season for all pea varieties in Perloja. However, in Dotnuva, the correlation among the same factors was considerably weaker. A significant strong correlation between the AUDPC values of ascochyta blight and the amount of precipitation in the second half of the growing season was established only for Profi and Pinochio. The correlation of the AUDPC values of ascochyta blight and the sum of effective temperatures was moderate for the same varieties. In Dotnuva, in the crops of other varieties, meteorological indicators correlated with ascochyta blight weakly or did not correlate at all. It is likely that such results were obtained because of the low severity of ascochyta blight in Dotnuva, compared with Perloja, where the disease severity was markedly higher. Ascochyta blight incidence on pods was significantly influenced both in Dotnuva and Perloja by the amount of precipitation in the second half of the growing season (r=0.80-0.99), and in Perloja also by effective temperatures, from r=0.70 to 0.87. The conidia of the pathogens of Ascochyta complex are dispersed by rain-splash onto other plants horizontally and upwards to the newly emerging plant parts (Roger, Tivoli, 1996; Schoeny et al., 2008). A minimum wet period of 2h is required for the germination of M. pinodes conidia at temperatures from 15 to 30 °C. The pathogenic conidia that have been dispersed to newly emerging plant parts can survive dry periods of up to 21 days and can successfully germinate after the rain. Roger et al. (1999) indicated that ascochyta blight infection was severe in each case when plants were returned to wet conditions after a period of dryness. Setti et al. (2008) reported that lengthening of the duration of wet period increased ascochyta blight severity in the crops of all pea varieties tested. Moreover, the authors maintain that a 48-hour period of uninterrupted wetness on plants in the crops susceptible to ascochyta blight stipulated the outbreak of infection and is an indicator for use of fungicides.

13 Table 4. The correlation coefficients (R) among the AUDPC values of pea root and foot rots and ascochyta blight and the amount of precipitation (mm) and the sum of effective temperatures (∑T≥5 °C). 2008–2010 Environmental indicators Profi Eiffel Simona Tinker Mascara Pinochio R between AUDPC of root rots and environmental indicators Dotnuva Precipitation 04.21-05.31 0.53 0.21 0.17 0.59 0.60 0.67* Precipitation 06.01-07.21 0.92** 0.97** 0.93** 0.95** 0.92** 0.93** ∑T≥5°C 04.21-05.31 0.46 0.77* 0.75* 0.44 0.40 0.35 ∑T≥5°C 06.01-07.21 0.81** 0.59 0.55 0.86** 0.86** 0.92** Perloja Precipitation 04.21-05.31 0.94** 0.99** 0.98** 0.98** 0.96** 0.92** Precipitation 06.01-07.21 0.08 0 0.08 0.18 0.27 0.23 ∑T≥5°C 04.21-05.31 0.45 0.54 0.60 0.67* 0.73* 0.32 ∑T≥5°C 06.01-07.21 0.56 0.65 0.7* 0.76* 0.81** 0.44 R between AUDPC of foot rots and environmental indicators Dotnuva Precipitation 04.21-05.31 0.71* 0.08 0.01 0.80** 0.47 0.52 Precipitation 06.01-07.21 0.29 0.84** 0.73* 0.83** 0.26 0.90** ∑T≥5°C 04.21-05.31 0.30 0.87** 0.72** 0.15 0.63 0.45 ∑T≥5°C 06.01-07.21 0.66 0.32 0.32 0.97** 0.23 0.79* Perloja Precipitation 04.21-05.31 0.92** 0.98** 0.97** 0.98** 0.96** 0.88** Precipitation 06.01-07.21 0.26 0.02 0.01 0.20 0.10 0.34 ∑T≥5°C 04.21-05.31 0.30 0.52 0.52 0.69* 0.52 0.21 ∑T≥5°C 06.01-07.21 0.42 0.63 0.63 0.78* 0.63 0.34 R between AUDPC of ascochyta blight and environmental indicators Dotnuva Precipitation 04.21-05.31 0.40 0.10 0.44 0.01 0.14 0.19 Precipitation 06.01-07.21 0.90** 0.56 0.09 0.62 0.46 0.92** ∑T≥5°C 04.21-05.31 0.54 0.46 0.44 0.55 0.55 0.73* ∑T≥5°C 06.01-07.21 0.70* 0.32 0.29 0.28 0.10 0.55 Perloja Precipitation 04.21-05.31 0.66 0.68* 0.56 0.73* 0.63 0.71* Precipitation 06.01-07.21 0.80** 0.79* 0.87** 0.69* 0.83** 0.73* ∑T≥5°C 04.21-05.31 0.91** 0.99** 0.99** 0.94** 0.99** 0.96** ∑T≥5°C 06.01-07.21 0.99** 0.99** 0.97** 0.96** 0.99** 0.97** R between ascochyta blight severity on pods and environmental indicators Dotnuva Precipitation 06.01-07.31 0.84** 0.98** 0.97** 0.80* 0.96** 0.88** ∑T≥5°C 06.01-07.31 0.47 0.48 0.49 0.43 0.46 0.47 Perloja Precipitation 06.01-07.31 0.99** 0.99** 0.99** 0.99** 0.97** 0.97** ∑T≥5°C 06.01-07.31 0.73* 0.74* 0.70* 0.75* 0.78* 0.87** Number of pairs:in 2008 n=9; in 2009 n=9; in 2010 n=9. *P≤ 0.05; **P≤ 0.01.

Frequency of detection of the pathogens of Ascochyta complex on field peas. Pathogens of the Ascochyta complex were abundantly detected on pea seedlings in both experimental sites. It is likely that the major source of pathogens isolated at an early growth stage was seed-borne infection. All pathogens of the Ascochyta complex are seed-borne; M. pinodes and P. pinodella can cause root and foot rots, while first 14 symptoms of A. pisi appear on a stemlet above the first leaves (Walen, 1965; Bretag ir kt., 2006). Later, with rapid growth of plants, until flowering stage, the frequency of detection of pathogens of Ascochyta complex diminished, and during flowering-grain formation stage the detection frequency rapidly increased until maturity stage (Table 5). Table 5. The frequency of detection of Ascochyta complex on peas at different plant growth stages (BBCH) in Dotnuva and Perloja Variety Frequency of detection (%) of Ascochyta complex 11-13 36-37 51 61-63 77-79 On pods 2009, Dotnuva Profi 24.0 16.0 10.0 16.7 60.0 73.3 Eiffel 40.0 70.0 3.3 43.3 60.0 60.0 Simona 52.0 32.0 23.3 50.0 50.0 53.3 Tinker 28.0 76.7 16.7 20.0 30.0 60.0 Mascara 64.0 53.3 33.3 30.0 30.0 66.7 Pinochio 28.0 43.3 30.0 16.7 60.0 73.3 2009, Perloja Profi 16.7 - - 83.3 73.3 - Eiffel 6.6 - - 43.3 80.0 - Simona 26.7 - - 73.3 80.0 - Tinker 10.0 - - 76.7 63.3 - Mascara 6.7 - - 46.7 66.3 - Pinochio 13.3 - - 66.7 60.0 - 2010, Dotnuva Profi 44.0 20.0 36.0 60.0 56.0 46.7 Eiffel 76.0 53.3 16.0 80.0 56.0 100 Simona 80.0 40.0 40.0 76.0 72.0 10.0 Tinker 60.0 73.3 12.0 68.0 80.0 60.0 Mascara 68.0 86.7 44.0 60.0 60.0 20.0 Pinochio 84.0 73.3 28.0 64.0 60.0 28.0 2010, Perloja Profi - 35.0 16.0 44.0 16.0 33.3 Eiffel 8.0 53.3 20.0 - 40.0 89.0 Simona 24.0 65.0 12.0 24.0 16.0 85.0 Tinker 60.0 84.0 52.0 28.0 64.0 39.0 Mascara 52.0 40.0 8.0 16.0 12.0 50.0 Pinochio 34.3 30.0 48.0 12.0 28.0 85.0

Both in Dotnuva and Perloja, the frequency of pathogen detection on individual varieties varied appreciably at different growth stages. Until grain-formation stage, only solitary isolates of A. pisi were detected. At maturity stage, the highest relative density of this pathogen in the Ascochyta complex was identified on the stems of Tinker and Mascara. The pods of all varieties were severely infected with the pathogens of Ascochyta complex, among which A. pisi took the dominant position. The effect of seed treatment on seed-borne infection of pea seedlings. After a 21-day incubation in wet blotter rolls, all untreated seeds in 2008, and in 2010 nearly 15 all untreated seeds exhibited brownish or black lesions, and in 2009 half of the seeds showed infection symptoms. In all experimental years, germination of untreated seed was lower than that of the seed treated with Raxil extra and Kinto (Figure 1).

100

80

60

40

20

0 Untreated Kinto Raxil extra Untreated Kinto Raxil extra Untreated Kinto Raxil extra

2008 2009 2010 Seedlings with hypocotyls affected % Seedlings with roots affected % Seeds affected % Germinated seeds %

Figure 1. The effect of seed treatment on pea seed germination and seed-borne seedling infection with rots in 2008-2010. Standard deviation bars are given.

Hypocotyls and rootlets of the seedlings grown from untreated seeds were severely affected by seed-borne infection every year; especially high seedling infestation was observed in 2010. Lower seed infestation in 2009 resulted in a smaller number of seedlings with hypocotyl and rootlet lesions. Fungicide treatment gave a very marked reduction of seedlings’ hypocotyl and rootlet infection with seed borne causal agents in each experimental year. Infected seed is the main source of initial infection of all the three pathogens of Ascochyta complex – M. pinodes, A. pisi, P. Pinodella. Moussart et al. (1998) indicated an especially high transmission of seed-born M. pinodes infection into seedlings, reduction of seed germination, seedling rots caused by the pathogen, and death of young plants. Hwang et al. (1991) have pointed out that soil moisture and temperature are of crucial importance for the transmission of seed-borne infection into a germinating seedling. Control of pea root and foot rots with seed treatment. The effect of seed treatment agents on the severity of root and foot rots was estimated by comparing the data from the treatments sown with untreated seed and Kinto and Raxil extra-treated seed. The incidence of pea root rots in 2008 and 2009 was higher in Perloja than in Dotnuva. In 2008 and 2009, root rots occurred on pea plants already at seedling stage (Fig. 2 A and B, D and E). However, in 2010 the severity of root rots at seedling stage was low both in Dotnuva and Perloja (Fig. 2 C, F). In 2008, in the plots sown with treated seed a significant reduction in root rot severity was observed to last the longest until grain- formation stage (BBCH 75-77); however, in 2009 and 2010 this effect lasted until flowering, except for the plots sown with Raxil extra-treated seed in Perloja (2009). The least seed treatment effect was recorded in 2010 in Perloja, while in Dotnuva, in the same year, a significant reduction in the severity of rots was observed until pea flowering stage. The first symptoms of foot rots, like those of root rots, were spotted already at seedling stage. With further plant development, the severity of foot rots, like that of root rots, increased both in the plots sown both with untreated and treated seed; however, the 16 disease progress curves differed between years and sites (Fig. 3). The highest level of foot rot severity at ripening stage was in 2009 in Perloja, and in 2010 both in Perloja and Dotnuva, while the least – in 2008 and 2009 in Dotnuva. Compared with the untreated, in the plots sown with treated seed the plants were significantly less affected by foot rots in most cases until flowering. However, in separate cases (in 2008 in Dotnuva) the severity of foot rots on plants grown from treated seed was significantly lower up until ripening stage.

100 100 A D 80 80

60 60

40 40

20 20 Disease severity index, % index, Disease severity 0 0 15 33-35 51-55 65-69 75-77 84-85 11 33-35 51-55 61 65-69 75-77 85

100 100 B E 80 80

60 60

40 40

20 20 Disease severity index, % index, Disease severity 0 0 13 36-37 54-55 61-62 79 84-85 13 36-37 40 61-62 71 77-79 83

100 100 C F 80 80

60 60

40 40

20 20

Disease severity index, % index, Disease severity 0 0 13 36-37 54-55 61-63 79 84-85 11 36 39 61 79 84-85 Growth stage Growth stage Fig. 2. Root disease development in 2008 (A), 2009 (B) and 2010 (C) in the untreated (◊), Kinto-treated (□) and Raxil extra-treated (∆) treatments in Perloja (left) and Dotnuva (right). Standard deviation bars are given.

Progress curves of root and foot rots during pea growing season differed between the experimental sites. Since spring, the severity of both root rots and foot rots was higher in Dotnuva, except for the exceptionally droughty year 2009, and in the second half of the growing season – in Perloja, although the amount of rainfall in the first half of the growing season was higher in Perloja in all experimental years. This might have been caused by different water holding capacity of the soils (Webb et al., 1993). Compared with sandy loams of Southeast Lithuania (Perloja site), heavier soils of Middle Lithuania (Dotnuva site) were better at preserving spring moisture, which created more conducive conditions to the occurrence of pathogen infection, while sandy soils with low moisture holding capacity dried up more rapidly before the crop had covered the soil surface. Later, in the second half of the growing season, after the plants had completely covered

17 the soil surface and heavy rain occurred, very favourable microclimate was created for the development of rots in Perloja.

100 100 A D 80 80

60 60

40 40

20 20 Disease Disease severity index, % 0 0 15 33-35 51-55 65-69 75-77 84-85 11 33-35 51-55 61 65-69 75-77 85

100 100 B E 80 80

60 60

40 40

20 20 Disease Disease severity index, % 0 0 13 36-37 54-55 61-62 79 84-85 13 36-37 40 61-62 71 77-79 83

100 100 C F 80 80

60 60 40 40 20 20 Disease index,severity % Disease 0 0 11 36 39 61 79 84-85 13 36-37 54-55 61-62 79 84-85 Growth stage Growth stage Fig. 3. Foot rot disease development in 2008 (A), 2009 (B) and 2010 (C) in the untreated (◊),Kinto-treated (□) and Raxil extra- treated (∆) treatments in Perloja (left) and Dotnuva (right). Standard deviation bars are given.

In most cases, the AUDPC values of foot and root rots in the plots sown with treated seed were significantly lower than those in the untreated plots (Table 6). Comparison of the AUDPC of both root and foot rots of seed-treated and untreated plots under different conditions, showed that in Dotnuva in 2008 and 2010 the AUDPC values were higher than those in Perloja. This is likely to have been caused by the higher root rots severity until pea bud formation-flowering stage. However, in 2009 the AUDPC of root and foot rots differed appreciably between the sites. Compared with the entire experimental period, that year the highest AUDPC of both root and foot rots was in Perloja, while the lowest in Dotnuva, where the development of rots was inhibited by droughty conditions that persisted from pea germination until flowering stage, and conversely, in Perloja unusually warm weather with heavy rains created conducive conditions to the occurrence of rot infection. Literature sources indicate that causal agents of pea root rots - Fusarium solani var. pisi and Pythium ultimum affect plants more severely on wet soils compared with the soils of normal moisture or dry soils. In a wet soil, a significant increase in the populations of root rots causal agents in the pea rhizosphere has been noted (Cook, Flentje, 1967; Kerr, 1964). Moreover, in the 18 conditions of elevated moisture, the germination of chlamidospores of Fusarium and other fungi increases, causing an increase in the amount of inoculums in the rhizosphere, which in turn triggers a severe infection of root rots. (Bhatti, Kraft, 1992). Table 6. The AUDPC of pea root and foot rots in Perloja and Dotnuva 2008 2009 2010 2008 2009 2010 Treatment AUDPC of root rots AUDPC of foot rots Perloja Untreated 3009 4015 2125 2782 3937 2058 Kinto 2034** 3100** 1755* 2115** 3136** 1694** Raxil extra 1909** 3683 1576** 1933** 3774 1506** Dotnuva Untreated 2958 2702 3236 3030 2161 3309 Kinto 2434* 1668** 2672** 2376** 1240** 2873 Raxil extra 2179** 1885** 2314** 2646 1465** 2402** *) difference from the untreated (treatment 1) P≤0.05; **) difference from the untreated P≤0.01

Ascochyta blight control in field pea using fungicides. In 2008, at the time of fungicide application there were no symptoms of ascochyta blight in pea crops in both experimental sites. In Perloja, the spread of the disease became more intensive at grain- formation stage, while in Dotnuva at ripening stage. In all the treatments applied with ascochyta blight control measures, such as seed treatment, application of fungicides or their combinations, the disease severity was significantly (P≤ 0.01) lower than in the untreated (Table 7). In Dotnuva, ascochyta blight did not spread on pods, and in Perloja, only single symptoms of the disease were observed in the plots not applied with fungicides. In 2009, in Perloja, aschochyta blight occurred at flowering stage. In the plots sown with treated seed and sprayed with fungicides at grain ripening stage, the disease severity was significantly lower than in the untreated/unsprayed. In Dotnuva, where ascochyta occurred at the end of grain-formation stage, a significant disease severity reduction was noted only for the treatments (in two out of three) sown with Raxil extra- treated seed. In the plots, not applied with fungicides at flowering stage, ascochyta blight in Dotnuva spread on the majority of pods, and in Perloja on all pods; however, the disease incidence was low. The pods of fungicide-sprayed plants were not affected. In the exceptionally rainy year of 2010, both in Perloja and Dotnuva the spread of ascochyta blight was particularly intensive. In Perloja, it started to manifest itself at the bud formation stage; at maturity ascochyta blight severity was significantly lower both on plants and pods in all the plots applied with the disease control measures. In Dotnuva, the trend towards disease severity reduction both on plants and pods was observed in all treatments; however, only the plots applied with Signum were significantly less affected. In the plots, sown with treated seed and spray-applied with fungicides, the AUDPC values of ascochyta blight in most cases were significantly lower than in the control (Table 8).

19 Table 7. The effect of fungicides and seed treatment on ascochyta blight severity on plants (R%) and pods (RA%) in the field pea crops in Dotnuva and Perloja in 2008-2010 Treatments 2008 2009 2010 R% RA% R% RA% R% RA% Perloja Untreated /unsprayed 5.7 0.5 20.0 1.5 40.0 3.5 Untreated/Signum 1.4** 0 13.1** 0 24.5** 1.9** Untreated /Bravo 1.8** 0 14.5** 0 30.0* 2.1** Kinto /unsprayed 2.3** 0.5 16.5* 0.9 31.4* 2.1** Raxil extra /unsprayed 2.2** 0.3 17.0* 1.1 29.4** 1.6** Kinto/Signum 1.5** 0 13.0** 0 33.0 1.9** Raxil extra /Signum 1.3** 0 12.8** 0 26.6** 1.6** Kinto /Bravo 1.3** 0 15.0** 0 31.2* 2.4** Raxil extra /Bravo 1.1** 0 14.7** 0 24.0** 1.7** Dotnuva Untreated/unsprayed 15.8 0 3.3 1.0 39.3 2.6 Untreated /Signum 0.8** 0 3.5 0 21.4** 1.7 Untreated /Bravo 0.8** 0 3.1 0 34.4 2.2 Kinto /unsprayed 7.8** 0 3.8 1.35 34.4 1.9 Raxil extra /unsprayed 13.1* 0 1.6* 0.8 37.9 1.8 Kinto/Signum 0.6** 0 2.7 0 23.8** 1.6 Raxil extra /Signum 0.6** 0 2.3 0 20.9** 2.1 Kinto /Bravo 0.4** 0 2.2 0 33.9 2.0 Raxil extra /Bravo 1.7** 0 1.9* 0 33.5 1.8 *) difference from the untreated (treatment 1) P≤0.05; **) difference from the untreated P≤0.01 Table 8. The effect of seed treatment and fungicides on the AUDPC values of ascochyta blight in field pea crops in Perloja and Dotnuva in 2008-2010. Treatment Perloja Dotnuva 2008 2009 2010 2008 2009 2010 Untreated/unsprayed 91 265 618 103 31 514 Signum 35** 204** 400** 6** 27 264** Bravo 31** 226 478* 5** 31 421* Signum/Kinto 31** 204** 534 5** 22 226** Signum/Raxil extra 21** 197** 426* 4** 22 185** Bravo/Kinto 20** 226 527 3** 21 383** Bravo/Raxil extra 17** 221* 382** 11** 19* 311** *) difference from the untreated (treatment 1) P≤0.05; **) difference from the untreated P≤0.01 Comparison of the AUDPC values between the plots sprayed with Signum and Bravo fungicides did not reveal any distinct difference; however, in Dotnuva, under the conditions of severe infection in 2010, the AUDPC values of ascochyta blight of Signum-applied plots were markedly lower than those of Bravo-applied plots. Summarised results of ascochyta blight control using fungicides enables us to maintain that when treated seed is sown and fungicides are spray-applied at the flowering stage, the severity of ascochyta blight in a pea crop is significantly reduced.

20 Seed treatment is also an effective measure to reduce ascochyta blight infection in pea crops. The effect of seed treatment on the frequency of detection of the pathogens of Ascochyta complex on pea plants and grain. In 2009, in Dotnuva and Perloja, the frequency of detection of pathogens of Ascochyta complex on the seedlings germinated from the untreated seed was 10%, and that on the seedlings emerged from treated seed was 5.0 and 3.3 %, respectively. A particularly high increase in the frequency of detection of these pathogens on the plants in the untreated seed treatments occurred at stem elongation stage. Seed treatment had an effect on the detection frequency of ascochyta blight causal agents at seedling-stem elongation stage, at later growth stages the frequency of detection of pathogens isolated on the plants both in the seed-treated and untreated plots was similar. In 2010, in Dotnuva, unlike in the previous year, the frequency of detection of Ascochyta complex fungi was very high since the seedling stage (Table 9). The frequency of detection of ascochyta blight causal agents on the seedlings in the untreated seed plots was 88%, while that in the treated seed plots was considerably lower 12 and 36%. Table 9. The effect of seed treatment on the variation of frequency of detection (FD %) of Ascochyta complex and Fusarium spp. pathogens on pea foot in Dotnuva in 2009 and 2010 and in Perloja in 2009. Treatment FD % on pea foot during the growing season (BBCH) 11-13 36-37 51 71 11-13 36-37 51 Dotnuva, 2009 Ascochyta complex Fusarium spp. Untreated 10.0 13.3 36.7 63.3 72.0 90.0 73.3 Kinto 5.0 4.0 33.3 53.3 16.0 68.0 63.3 Raxil extra 5.0 3.3 16.7 66.7 52.0 70.0 63.3 Perloja, 2009 Ascochyta complex Fusarium spp. Untreated 10.0 60.0 16.7 66.7 60.0 50.0 73.3 Kinto 3.3 13.3 23.3 73.3 73.3 73.3 53.3 Raxil extra 3.3 6.7 13.3 73.3 36.7 33.3 86.7 Dotnuva, 2010 Ascochyta complex Fusarium spp. Untreated 88.0 60.0 44.0 64.0 52.0 20.0 68.0 Kinto 12.0 73.3 16.0 52.0 24.0 40.0 52.0 Raxil extra 36.0 60.0 44.0 56.0 56.0 50.0 48.0

Such contrast in the detection frequency of Ascochyta complex fungi between the years 2009 and 2010 was determined by the completely different meteorological conditions – in 2009, the weather was extremely droughty since early spring, while in 2010 in May there were as many as 15 rainy days, and the monthly rainfall amount was 80% greater than the long-term average. The frequency of detection of Fusarium fungi was high since the seedling stage. In Perloja, a lower detection frequency of these fungi at the seedling - stem elongation stage was identified on the seedlings emerged from the Raxil extra -treated seeds. In 21 Dotnuva, the detection frequency of Fusarium spp. fungi was high on the plants both in untreated and Raxil extra- seed treated plots, only in the Kinto seed-treated plots at seedling stage the detection frequency of Fusarium fungi was significantly lower. Since grain formation stage, detection frequency of Fusarium fungi declined. It is likely that because of the competition, which resulted in a marked increase in Ascochyta complex fungi, detection frequency of Fusarium spp. diminished. The frequency of detection of Ascochyta complex on the pods and grain of plants spray-applied with fungicides at flowering stage was highly dependent on the year and ascochyta blight severity in the experimental sites (Table 10). Table 10. The effect of fungicides on the frequency of detection (FD%) of pathogens of Ascochyta complex and relative density (RD%) of A. pisi in Ascochyta complex on pea pods and grain in Dotnuva and Perloja in 2008-2010. Year Fungicides Control Signum Bravo Untreated Signum Bravo Indicators Dotnuva Perloja On pods 2008 A. compl.FD% 7.0 10.0 3.0 - - - A. pisi RD% 100 100 30.0 - - - 2009 A .compl. FD% 51.1 82.0 91.1 100 100 100 A. pisi RD % 13.1 22.0 12.2 40.0 14.0 15.6 2010 A. compl. FD% 86.0 72.0 58.0 93.3 80.0 80.0 A. pisi RD % 68.6 66.7 65.5 14.3 25.0 0 On grain 2008 A. compl. FD% 0 0 0 78.0 27.0 38.0 A. pisi RD % 0 0 0 100 77.8 18.4 2009 A. compl. FD% 1.0 0 0 76.0 60.0 100 A. pisi RD % 100 0 0 40.0 28.0 52.0 2010 A. compl. FD% 9.0 2.0 10.0 31.0 8.0 0 A. pisi RD % 88.9 50.0 100 0 100 0

In Dotnuva, where ascochyta blight incidence was low in 2009, the grains were not infected with Ascochyta complex, although the frequency of detection of the pathogens was rather high on pods. With higher incidence of ascochyta blight, which was the case both in Dotnuva and Perloja in 2008 and 2010, an increase in the frequency of detection of Ascochyta complex on pods and grain was noted. At severe incidence of ascochyta blight, the grains of the fungicide spray-applied plants were less infected with the pathogens of Ascochyta complex in two years out of three, compared with the grain from the unsprayed plots. The frequency of detection of pathogen species of Ascochyta complex differed between experimental years: in 2008, in both experimental sites the prevalent species was A. pisi, in 2009 A. pisi accounted for nearly half of the isolated pathogens of Ascochyta complex; in 2010, in Dotnuva, A. pisi accounted for the larger half of the population, whereas in Perloja the prevalent species were M. pinodes and P. pinodella. Our research evidenced that in separate years the infection pressure from the pathogens of Ascochyta complex can be high on pea grains. The effect of seed treatment and fungicide application on field pea grain yield and yield components. Until flowering stage, the roots and foot of the pea plants in the seed-treated plots were less affected by rots, while fungicides provided a good 22 suppression of ascochyta blight severity on plants, therefore the yield increases resulting from the application of these measures were inconsistent (Table 11). This is likely to have been caused by a combination of the narrow experimental plots and showery weather: at the edges of the narrow plots pea plants lodged on the paths and in rainy conditions with heavy downpours in 2009 in Perloja and in 2010 in both experimental sites, the pea plants were badly lodged, which made their harvesting complicated. A thousand grain weight (TGW) of peas in Perloja in 2008 and 2009 was significantly higher in two out of three plots sprayed with the fungicide Signum, and in 2010 in all Signum-sprayed plots and in two out of three Bravo-sprayed plots. In Dotnuva, in 2008 a trend towards increasing of TGW was noted. In 2009, the increase was significant in the plots of all fungicide treatments, and in 2010 it was significantly higher in two out of three Signum-sprayed treatment plots. Table 11. The effect of seed treatment and fungicide application on pea grain yield in Perloja and Dotnuva in 2008 – 2010. Grain yield t ha-1 Treatment Perloja Dotnuva 2008 2009 2010 2008 2009 2010 Untreated/unsprayed 2.25 1.68 1.80 2.52 2.07 2.63 Untreated /Signum 2.55 1.73 2.21** 2.15 2.17 2.77 Untreated /Bravo 2.73 1.57 1.43 2.31 2.29* 3.04* Kinto /unsprayed 2.32 1.51 1.74 2.42 2.16 2.41 Raxil extra /unsprayed 2.42 1.55 2.16* 2.52 2.05 2.40 Kinto/Signum 2.46 1.94 1.86 2.45 2.35** 2.51 Raxil extra /Signum 2.47 1.73 1.64 2.38 2.22 2.27 Kinto /Bravo 2.84 1.45 1.52 2.26 2.38** 2.76 Raxil extra /Bravo 3.04* 1.66 2.40** 2.45 2.18 2.38 *) difference from the untreated (treatment 1) P≤0.05; **) difference from the untreated P≤0.01

Comparison of the two years’ data on pod and grain number and weight per plant (2008 and 2010), a trend was noticed, suggesting that in the seed-treated plots applied with fungicides the values of these indicators in most cases were higher than in the untreated/unspryed, and in 2010 in Dotnuva, in two out of three Raxil extra – seed treated and fungicide applied treatment plots pod number per plant was significantly higher than that in the untreated/unspryed. In the plots sown with only Raxil extra-treated seed there was also noted a trend for increasing of pod number. Also, in 2010, in Dotnuva, in the plots of all treatments of Raxil extra-treated seed, at flowering applied with fungicides and in the plots not applied with fungicides, as well as in Kinto/Bravo plots there was established a significantly higher grain number and weight per plant. Pea grain yield and its components in relation to the severity of root and foot rots and ascochyta blight. During the whole experimental period (2008-2010), no significant correlation was found between pea productivity and root and foot rot and ascochyta blight AUDPC either in Dotnuva or Perloja. In 2008-2010, in Dotnuva, we established a significant negative correlation of TGW with the AUDPC of root rots r=-0.63; -0.85; -0.60, with the AUDPC of foot rots in 2009 in Dotnuva r=-0.92, however no consistent correlation was determined between TGW and AUDPC of ascochyta blight. Negative moderate or strong significant correlation between pod and

23 grain number per plant, grain weight per plant and AUDPC of root and foot rots was established in 2010 in both experimental sites. A significant (P≤ 0.01) moderate negative correlation between these indicators and AUDPC of ascochyta blight was established only in one year 2008, r=-0.52; -0.53; -0.57, respectively. Numerous studies done in other countries have also indicated that treatment of seed infected with ascochyta blight causal agents improved germination under field conditions; however, the seed treatment did not give any grain yield increase (Hwang ir kt., 1991; Biddle, 1994; Xue, 2000). Conversely, the study done by Gorfu and Sanghote (2003) evidenced that seed treatment with Carbendazim resulted in a 13.2% pea grain yield increase, and Iprodione gave a yield increase of 12.5%, compared with the untreated control. Xue et al. (2003) have reported that the spray-application of the crops of 10 pea varieties with Chlorothalonil provided a significant reduction in ascochyta blight pressure, an increase in grain yield and TGW in all the three experimental years; however, the variety x fungicide interaction was not significant in all years. The yield increase resulting from the fungicide application amounted to on average 6.4%, and TGW increase was 0.9-5.1%. Moreover, the pea seed treated with Chlorothalonil was 19.2 % less infected with the propagules of M. pinodes. Tivoli et al. (1996) found that severe ascochyta blight infection can reduce seed number per plant, depending on the year’s infection pressure, by 18 and 25% and seed size by 13.5 and 16.7%, and give a seed yield reduction as high as 40%, compared with healthy plants. In our study, we did not obtain such a difference, since even in fungicide-spray applied plots the plants were affected by ascochyta blight, although the disease severity was significantly lower. Side effect of chemical seed treatment on the biota of pea rhizosphere and soil. The results from the 2008-2010 period indicated that the applied pea seed treatment products provided a lasting effect on the total amount of micro-organisms both in the soil between rows (bulk soil) and in the pea rhizosphere. Seed treatment effect gave a significant reduction in the counts of fungal colony forming units (cfu g-1) both in the bulk soil and pea root rhizosphere (Fig. 4). 3 -1 cfu x10 g dw Fungi 80

60

40

20

0 BBCH 11-12 BBCH 39 BBCH 11-12 BBCH 39 BBCH 11-12 BBCH 39 2008 2009 2010

Rhizosphere untreated Rhizosphere Raxil extra Rhizosphere Kinto Bulk soil untreated Bulk soil Raxil extra Bulk soil Kinto Figure 4. The effect of seed treatment with Raxil extra and Kinto on the abundance of soil fungal population (cfu 103 g-1 dry soil) in the rhizosphere and soil. Standard deviation bar is indicated. At pea seedling stage, in all the three experimental years, the rhizosphere of plants in the seed-treated plots contained a lower total number of fungi: in 2008, at seedling 24 stage Raxil extra and Kinto reduced the fungal population by 41 and 33%, in 2009 by 33.8 and 24.5%, and in 2010 by 58.2 and 36.1%, respectively. Also, in two years (2008 and 2010) out of three, there was established a negative effect of seed treatment on the abundance of fungal population in the soil between pea rows (in bulk soil) – the amount of fungi in the soil near the seedlings in the seed-treated plots in 2008 was by on average 11.6%, and in 2010 by 25.8% lower, compared with the plots sown with untreated seed. At pea bud formation stage, the negative effect of seed treatment on the fungal population in the pea rhizosphere was still persistent – in the rhizosphere of plants in the seed-treated plots in 2008 the fungal counts were lower by 42.9%, and in 2010 by 19.3%. Our research results evidenced that with a decline in fungal population, an increase occurs in the soil bacterial population. CONCLUSIONS The study, aimed to investigate the severity of root and foot rots and ascochyta blight in field pea crops and to identify prevention and control measures of the diseases caused by the pathogens of the Ascochyta complex, done at the Research Centre for Agriculture and Forestry’s Institute of Agriculture in Dotnuva (Middle Lowland of Lithuania) and Perloja Experimental Station in Perloja (Southeast of Lithuania) over the period 2008-2010, generated the following conclusions: 1. In all experimental years, the highest susceptibility to root and foot rots in Dotnuva was exhibited by the pea variety Mascara, while in Perloja by the varieties Tinker and Mascara, the pea varieties Pinochio and Profi were found to be less susceptible. Irrespective of the ascochyta blight infection level, Tinker demonstrated the highest susceptibility to the disease, while Simona and Pinochio were less susceptible. 2. The amount of precipitation during the growing season and the sum of effective temperatures (∑≥ 5 °C) exerted a direct influence on the AUDPC values of pea root and foot rots. In Dotnuva, a very strong positive correlation was established between the AUDPC values of pea root and foot rots and the amount of precipitation in the second half of the growing season, whereas in Perloja the AUDPC values of pea root and foot rots were strongly correlated with the amount of precipitation in the first half of the growing season. In both experimental sites, a strong correlation between the AUDPC values of root rots and the sum of effective temperatures was established for Tinker and Mascara, while the AUDPC values of foot rots strongly correlated with the sum of effective temperatures for Eiffel, Simona and Tinker varieties. 3. In Perloja, a significant moderate or strong correlation was identified between the AUDPC values of pea ascochyta blight and amount of precipitation and sum of effective temperatures for all field pea varieties tested. Due to the low severity of ascochyta blight in Dotnuvoje, the interaction among the same factors was markedly lower. The incidence of ascochyta blight on pods was significantly influenced by the amount of precipitation in the second half of the growing season (from r=0.80 to r=0.98 and r=0.97 to r=0.99 for individual varieties, respectively) in both experimental sites, while only in Perloja it was also significantly influenced by active temperatures (from r=0.70 to r=0.87). 4. The frequency of detection of pathogens of Ascochyta complex was high at pea seedling stage, later, until flowering, the frequency of pathogen detection diminished, and during the flowering–grain formation stage it increased again. In Dotnuva, at

25 seedling stage and during the growing season Mycosphaerella pinodes and Phoma pinodella patogens prevailed in the Ascochyta complex, and at the end of the growing season, Ascochyta pisi was prevalent on pods. In Perloja, the relative density of A. pisi in the population of Ascochyta complex was low during the entire growing season. There was found no consistent relationship between the frequency of detection of pathogens and pea variety. 5. Pea seedlings were severely affected by rots resulting from the seed-borne infection. Seed treatment with Raxil extra (a.i. tebuconazole+thiram 15+500 g l-1) and Kinto (a.i. triticonazole+prochloraz 20+60 g l-1) provided a significant reduction in the severity of root and foot rots only until flowering stage, and in some exceptional cases until grain formation stage. 6. Ascochyta blight severity was significantly lower in the plots sown with Raxil extra and Kinto-treated seed and / or applied with the fungicides Signum (a.i. boscalid+pyraclostrobin 267+67 g kg-1) and Bravo (a.i. chlorothalonil 500 g l-1) at flowering stage. The AUDPC values of ascochyta blight in the fungicide-applied plots in most cases were significantly lower in both experimental sites. 7. The frequency of detection of the pathogens of Ascochyta complex on the foot of pea plants in the seed-treated plots in the year 2009, which was less conducive to the occurrence of ascochyta blight, was significantly lower until flowering stage, and in the 2010, which was particularly favourable for the disease – until stem elongation stage. 8. The frequency of detection of the pathogens of Ascochyta complex on pea pods and grain depended on the level of ascochyta blight incidence. With rising infection level, the frequency of detection of pathogens increased both on pods and grain. In Perloja, the fungi of Ascochyta complex on grain were detected in all experimental years, while in Dotnuva only in 2010. The grains of pea crops spray-applied with the fungicides Signum and Bravo in two years out of three were less infected with the pathogens of Ascochyta complex, compared with those from the unsprayed plots. 9. Pea grain yield increments resulting from the use of seed treatment and fungicide application were inconsistent in both experimental sites. It was noted that the values of productivity indicators, TGW and pod number per plant were higher for the Raxil extra seed-treated plots spray-applied with the fungicides Signum and Bravo; in separate years these values significantly differed from the untreated/unsprayed. 10. In Dotnuva, in 2008-2010, a significant negative correlation was established between the TGW and AUDPC of root rots r= -0.63; -0.85; -0.60, respectively, and in 2009, between TGW and AUDPC values of foot rots (r= -0.92). In 2010, a significant negative correlation was established between pod and grain number per plant, grain weight per plant and AUDPC of root rots r= -0.59; -0.83; -0.84, respectively, and the same indicators and AUDPC of foot rots r= -0.61; -0.75; -0.71, respectively. In Perloja, no correlation was established between the AUDPC values of root and foot rots and yield indicators. 11. In Perloja, in 2008, a significant negative correlation was established between the AUDPC values of ascochyta blight and pod and grain number per plant and grain weight per plant r= -0.52; -0.53; -0.57, and TGW (r= -0.45). Low correlation coefficients were obtained in Dotnuva because the ascochyta blight infection level was very low there.

26 12. Seed treatment exerted a negative effect on soil fungal population. A particularly marked reduction in the counts of soil fungi in the rhizosphere of seedlings was identified in all experimental years; however, with the reduction in fungal population, an increase in soil bacteria population was observed. List of publications on the subject of the dissertation An articles in reviewed periodical publications: Gaurilčikienė I, Česnulevičienė R., Janušauskaitė D., Ronis A. Management of seed borne root rot diseases of pea (Pisum sativum L.) with a fungicide seed treatment // Žemdirbystė=Agriculture. – 2012. vol. 99, No. 1, p. 80–88. Cittation index: 0.232 Gaurilčikienė I., Janušauskaitė D., Česnulevičienė R., Ramanauskienė J. The suppression of stem base and root rot diseases of pea as affected by fungicidal seed treatment // Žemdirbystė=Agriculture. – 2008, vol. 95, No. 3, p. 50–57. Articles/abstract in conference proceedings: Česnulevičienė R., Gaurilčikienė I., Janušauskaitė D. Pusiau belapių žirnių grybinių ligų epidemiologiniai tyrimai, jų prevencija ir kontrolinės priemonės // Agrariniai ir miškininkystės mokslai: naujausi tyrimų rezultatai ir inovatyvūs sprendimai Nr. 2. – Akademija, Lietuva, 2011, p. 45–46. Gaurilčikienė I., Česnulevičienė R. The effects of seed treatment on Ascochyta blight infection progress in pea in different agro ecosystems. Seed Health in Agricultural Development: Proceedings of 3rd International Symposium on SHAD. Ed. Jian-Qiang Li et al. – Benjing, China, 2010, p. 88–89. Česnulevičienė R., Gaurilčikienė I., Janušauskaitė D. Grybinių ligų išplitimo žirnių pasėliuose priklausomybė nuo genotipo bei agroekosistemose // Dirvožemio stabilumo užtikrinimas ekologiškai ir socialiai jautriuose regionuose. – Perloja, Lietuva, 2009, p. 171–177. Gaurilčikienė I., Janušauskaitė D., Česnulevičienė R., Ramanauskienė J. The Suppression of stem base and root rot diseases of pea as affected by fungicidal seed treatment / Advances in plant protection strategies: international plant protection conference. – Druskininkai, Lithuania, 2008, p. 27. Oral/poster presentations at conferences: Presentation (Oral) “The epidemiology of fungal diseases of semi-leafless pea, their prevention and control” at the conference “Agricultural and forestry sciences: recent research results and innovative solutions”, Akademija, 22–25 of February, 2011. Presentation (Poster) „The effects of seed treatment on ascochyta blight infection progress in pea in different agro ecosystems” at the international symposium „Seed Health and Agricultural Development“, Beijing, China, 25–27 of August, 2010. Presentation (Oral) “The development of fungal diseases of pea in dependence on genotype and agro ecosystem” at the international conference “Soil stability in ecologically and socially vulnerable regions”, Perloja, Lithuania, 10–12 of June, 2009. Presentation (Poster) “The suppression of stem base and root rot diseases of pea as affected by fungicidal seed treatment” at the international conference “Advances in plant protection strategies” Druskininkai, Lithuania, 10–12 September 2008. Recommendations for agriculture: Gaurilčikienė, R. Česnulevičienė, D. Janušauskaitė Root rot and Ascochyta blight control in pea // Naujausios rekomendacijos žemės ir miškų ūkiai. – 2011, p. 29–30

27 REZIUMĖ Sėjamojo žirnio (Pisum sativum L.) grybinių ligų žalingumo, jų prevencijos ir kontrolės tyrimai atlikti 2008 - 2011 metais Lietuvos agrarinių ir miškų mokslų centro filialuose Žemdirbystės institute ir Perlojos bandymų stotyje 2008-2010 m. Tyrimų hipotezė. Šaknų, pašaknio puvinių ir askochitozės išplitimas bei žalingumas priklauso nuo žirnio genotipo ir agroekologinių veiksnių. Apdorojant žirnių sėklą beicais galima sumažinti šaknų pašaknio puvinių intensyvumą. Purškiant žirnių pasėlius žydėjimo tarpsniu fungicidais tikėtina, kad galima sumažinti askochitozės žalingumą ir padidinti žirnių derlingumą. Tyrimų tikslas ir uždaviniai. Tyrimais siekta ištirti šaknų, pašaknio puvinių ir askochitozės išplitimą bei žalingumą sėjamojo žirnio pasėliuose, nustatyti Ascochyta komplekso patogenų sukeliamų ligų prevencijos ir kontrolės priemones. Tyrimų uždaviniai: - Nustatyti įvairių sėjamojo žirnio veislių jautrumą šaknų, pašaknio puviniams ir askochitozei skirtingomis agroekologinėmis sąlygomis. - Nustatyti meteorologinių faktorių įtaką šaknų, pašaknio puvinių ir askochitozės intensyvumui žirniuose. - Nustatyti Ascochyta komplekso patogenų aptikimo dažnį ant įvairių veislių žirnių. - Įvertinti Ascochyta komplekso patogenų sukeliamų ligų kontrolės galimybę naudojant beicus ir fungicidus. - Įvertinti beicų ir fungicidų įtaką žirnių derlingumui ir derliaus komponentams. - Ištirti galimą cheminių beicų šalutinį poveikį žirnių rizosferos bei dirvožemio mikroflorai. Mokslinis darbo naujumas. Pirmą kartą Lietuvoje ištirtas sėjamojo žirnio šaknų, pašaknio puvinių ir askochitozės intensyvumo raidos priklausomumas nuo genotipo ir meteorologinių veiksnių. Nustatyta iki šiol nenagrinėtas Ascochyta komplekso patogenų aptikimo dažnis ant pusiau belapių žirnių įvairiais augalų ontogenezės etapais. Ištirtas šių patogenų sukeltų ligų kontrolės veiksmingumas, apdorojant sėklą beicais, pasėlius – fungicidais bei naudotų priemonių įtaka žirnių derlingumui ir derliaus komponentams. Nustatytas cheminių beicų šalutinis neigiamas poveikis žirnių rizosferos ir dirvožemio mikrogrybams bei teigiamas – bakterijoms. Praktinė darbo reikšmė. Tyrimais nustatytos mažiau jautrios šaknų, pašaknio puviniams ir askochitozei sėjamojo žirnio veislės, kurios gali būti auginamos tausojančios ir ekologinės žemdirbystės sistemų ūkiuose. Ištirtos Ascochyta komplekso sukeltų ligų kontrolės priemonės. Sėklos beicavimas sumažino šaknų ir pašaknio intensyvumą iki žydėjimo tarpsnio. Pasėlių purškimas fungicidais stabdė askochitozės išplitimą ant ankščių ir grūdų. Įdiegtos praktikoje Ascochyta komplekso prevencijos ir kontrolės priemonės leis sumažinti žirnių grūdų derliaus nuostolius. Disertacijos ginamieji teiginiai: - Sėjamojo žirnio veislių jautrumas šaknų, pašaknio puviniams ir askochitozei priklauso nuo genotipo ir agroekologinių sąlygų. - Meteorologinės sąlygos daro ženklią įtaką šaknų, pašaknio puvinių ir askochitozės intensyvumui žirniuose. - Ascochyta komplekso patogenai aptinkami ant žirnių nuo daigų tarpsnio ir vegetacijos metu infekuoja visą augalą.

28 - Ascochyta komplekso patogenų sukeliamų ligų žalos mažinimas galimas apsaugant daigus nuo sėklos infekcijos ir purškiant pasėlius fungicidais. - Sėklos beicavimas ir pasėlių purškimas fungicidais didina žirnių derlingumą. - Cheminių beicų naudojimas sėklos apdorojimui gali turėti šalutinį neigiamą poveikį žirnių rizosferos ir dirvožemio mikroorganizmams. Tyrimų rezultatų aprobavimas. Pagrindiniai tyrimų duomenys paskelbti 2 moksliniuose straipsniuose, leidiniuose, referuojamuose ISI WOS duomenų bazėje, 4 straipsniuose, išspausdintuose konferencijų leidiniuose bei paruošta viena rekomendacija žemdirbiams. Tyrimų rezultatai buvo pateikti tarptautinėse konferencijose „Advances in plant protection strategies“ (Druskininkai, 2008 m., stendinis pranešimas), ,,Dirvožemio stabilumo užtikrinimas ekologiškai ir socialiai jautriuose regionuose“ (Perloja, 2009 m., žodinis pranešimas), tarptautiniame simpoziume „Seed Health in Agricultural Development“ (Pekinas, Kinija, 2010 m., stendinis pranešimas), konferencijoje „Agrariniai ir miškininkystės mokslai: naujausi tyrimų rezultatai ir inovatyvūs sprendimai“ (Akademija, 2011, žodinis pranešimas). Disertacijos turinys ir apimtis. Disertaciją sudaro įvadas, literatūros apžvalga, tyrimo metodų ir sąlygų pristatymas, tyrimų rezultatai ir jų aptarimas, išvados, darbe naudotos literatūros sąrašas bei disertantės su bendraautoriais paskelbtų publikacijų sąrašas. Darbo rezultatai pateikti 36 lentelėse ir iliustruoti 18 paveikslų. Panaudota 246 literatūros šaltiniai. Darbo apimtis 101 puslapis. IŠVADOS Apibendrinant Lietuvos agrarinių ir miškų mokslo centro filialuose Žemdirbystės institute, Dotnuvoje (Vidurio žemuma) ir Perlojos bandymų stotyje, Perlojoje (Pietryčių aukštuma) 2008-2010 m. atliktus tyrimus, kuriais siekta ištirti sėjamojo žirnio šaknų, pašaknio puvinių ir askochitozės žalingumą skirtingomis agroekologinėmis sąlygomis, nustatyti Ascochyta komplekso patogenų sukeltų ligų prevencijos ir kontrolės priemones, buvo padarytos šios išvados: 1. Visais tyrimo metais jautriausi šaknų ir pašaknio puviniams Dotnuvoje buvo Mascara, Perlojoje – Tinker ir Mascara veislių žirniai. Mažiau jautrūs puviniams buvo Pinochio ir Profi. Nepriklausomai nuo askochitozės infekcijos smarkumo jautriausi ligai buvo Tinker, o mažiau jautrūs buvo Simona ir Pinochio veislių žirniai. 2. Vegetacijos periodo kritulių kiekis ir efektyvių temperatūrų suma (∑≥ 5°C) turėjo tiesioginę įtaką žirnių šaknų ir pašaknio puvinių AUDPC reikšmėms. Dotnuvoje labai stiprus teigiamas koreliacinis ryšys buvo tarp žirnių šaknų ir pašaknio puvinių AUDPC reikšmių ir kritulių kiekio antroje, o Perlojoje tarp tų pačių AUDPC reikšmių ir kritulių kiekio pirmoje vegetacijos laikotarpio pusėje. Abiejose tyrimo vietose stipri šaknų puvinių AUDPC reikšmių koreliacija su efektyvių temperatūrų suma nustatyta Tinker ir Mascara, o pašaknio AUDPC – Eiffel, Simona ir Tinker veislių žirnių. 3. Perlojoje nustatytas esminis vidutinis arba stiprus koreliacinis ryšys tarp žirnių askochitozės AUDPC reikšmių ir kritulių kiekio bei efektyvių temperatūrų sumos visose žirnių veislėse. Dėl nedidelio askochitozės intensyvumo Dotnuvoje tų pačių faktorių sąveika buvo gerokai silpnesnė. Askochitozės išplitimą ant ankščių iš esmės ir Dotnuvoje, ir Perlojoje lėmė kritulių kiekis antroje vegetacijos pusėje (atitinkamai skirtingose veislėse nuo r=0,80 iki r=0,98 ir r=0,97 iki r=0,99), o Perlojoje ir aktyvios temperatūros (nuo r=0,70 iki r=0,87). 29 4. Ascochyta komplekso patogenai ant žirnių buvo gausiai aptinkami daigų tarpsniu, vėliau iki žydėjimo patogenų aptikimo dažnis mažėjo, o žydėjimo – grūdo formavimosi tarpsniu vėl padidėjo. Daigų tarpsniu ir vegetacijos metu Dotnuvoje Ascochyta komplekse vyravo Mycosphaerella pinodes ir Phoma pinodella patogenų kompleksas, o vegetacijos pabaigoje ant ankščių – Ascochyta pisi. Perlojoje A. pisi santykinis tankis Ascochyta komplekso populiacijoje buvo mažas visos vegetacijos metu. Nuoseklios priklausomybės patogenų aptikimo dažnio nuo žirnių veislės nenustatyta. 5. Žirnių daigai buvo smarkiai pažeisti puvinių nuo sėklos infekcijos. Apdorojant žirnių sėklą beicais tebukonazolu 15 g l-1+tiramu 500 g l-1 (Raksil ekstra) ir tritikonazolu 20 g l-1 +prochlorazu 60 g l-1 (Kinto), esminis šaknų ir pašaknio puvinių intensyvumo sumažėjimas stebėtas iki žydėjimo tarpsnio, o išimtinais atvejais iki grūdo formavimosi tarpsnio. 6. Žirnių askochitozės intensyvumas iš esmės buvo mažesnis beicais tebukonazolu 15 g l-1 + tiramu 500 g l-1 ir tritikonazolu 20 g l-1+prochlorazu 60 g l-1 apdorota sėkla sėtuose ir / ar fungicidais boskalidu 267 g kg-1+piraklostrobinu 67 g kg-1 (Signum) ir chlorotalonilu 500 g l-1 (Bravo) augalų žydėjimo tarpsniu nupurkštuose laukeliuose. Askochitozės AUDPC reikšmės fungicidais apdorotuose laukeliuose daugumoje atvejų buvo iš esmės mažesnės ir Dotnuvoje, ir Perlojoje. 7. Ant beicais apdorota sėkla sėtų žirnių pašaknio Ascochyta komplekso patogenų aptikimo dažnis mažiau palankiais askochitozei plisti 2009 m. buvo ženkliai mažesnis iki žirnių žydėjimo tarpsnio, o ypatingai palankiais 2010 m. – iki stiebo augimo tarpsnio. 8. Ascochyta komplekso patogenų aptikimo dažnis ant augalų ankščių ir grūdų priklausė nuo askochitozės išplitimo intensyvumo. Infekcijai smarkėjant, patogenų aptikimo dažnis didėjo ir ant ankščių, ir ant grūdų. Perlojoje grūdai Ascochyta komplekso grybais buvo infekuoti visais metais, o Dotnuvoje – tik 2010 m. Fungicidais boskalidu 267 g kg-1+ piraklostrobinu 67 g kg-1 ir chlorotalonilu 500 g l-1 purkštų augalų grūdai du metus iš trijų buvo mažiau užkrėsti Ascochyta komplekso patogenais nei nepurkšti. 9. Žirnių grūdų derliaus priedai nuo beicų ir fungicidų panaudojimo buvo nenuoseklūs tiek Dotnuvoje, tiek Perlojoje. Pastebėta, kad beicu tebukonazolu 15 g l-1 + tiramu 500 g l-1 apdorota sėkla sėtų ir fungicidais boskalidu 267 g kg-1+ piraklostrobinu 67 g kg-1 ir chlorotalonilu 500 g l-1 purkštų žirnių tiek derlingumo, tiek TGS bei ankščių skaičiaus ant augalo rodikliai buvo didesni, o atskirais metais ir iš esmės skyrėsi nuo kontrolės. 10. Dotnuvoje 2008-2010 m. nustatyta esminė neigiama koreliacija tarp TGS ir šaknų puvinių AUDPC, atitinkamai r= -0,63; -0,85; -0,60, bei 2009 m. tarp TGS ir pašaknio puvinių AUDPC reikšmių (r= -0,92). 2010 m. nustatytas esminis neigiamas koreliacinis ryšys tarp ankščių ir grūdų skaičiaus ant augalo, vieno augalo grūdų svorio ir šaknų puvinių AUDPC, atitinkamai r= -0,59; -0,83; -0,84, bei tų pačių rodiklių ir pašaknio puvinių AUDPC, atitinkamai r= -0,61; -0,75; -0,71. Perlojoje tarp šaknų, pašaknio puvinių AUDPC reikšmių ir derliaus rodiklių koreliacijos nenustatyta. 11. Perlojoje 2008 m. nustatyta askochitozės AUDPC reikšmių esminė neigiama koreliacija su ankščių ir grūdų skaičiumi ant augalo, vieno augalo grūdų svoriu, atitinkamai r= -0,52; -0,53; -0,57, bei TGS (r= -0,45). Dotnuvoje gauti maži koreliacijos koeficientai dėl to, kad ten buvo labai mažas askochitozės infekcijos lygis. 12. Beicai, naudoti žirnių sėklos apdorojimui, turėjo neigiamą poveikį dirvožemio grybų populiacijai. Ypač ženklus dirvožemio mikromicetų sumažėjimas daigų rizosferoje nustatytas visais tyrimų metais, tačiau sumažėjus grybų populiacijai stebėtas dirvožemio bakterijų populiacijos pagausėjimas. 30