A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 265 The contamination of Fabaceae plants with fungi and mycotoxins Aleksandra S. Orina1, Olga P. Gavrilova1, Tatiana Yu. Gagkaeva1, Aleksey A. Burkin2 and Galina P. Kononenko2 1Laboratory of Mycology and Phytopathology, All-Russian Institute of Plant Protection (VIZR), 196608 St. Petersburg, Pushkin, Podbelskogo sh. 3, Russia 2Mycotoxicology Laboratory, All-Russian Research Institute of Veterinary Sanitation, Hygiene, and Ecology – Skryabin and Kovalenko Federal Scientific Center, All-Russian Research Institute of Experimental Veterinary Medicine, 123022 Moscow, Zvenigorodskoe sh. 5, Russia e-mail: t.gagkaeva@yahoo.com This study aimed to assess the contamination of Fabaceae plants with fungi and mycotoxins, which have a negative effect on the health of consumers of plant products. Quantitative PCR was used to analyse the DNA of Alternaria, Cladosporium and Fusarium fungi in 69 plant samples harvested from four Russian regions in 2015. Additionally, mycotoxins were analysed using ELISA. Cladosporium fungi DNA was found in all the analysed samples; the occur- rence of Alternaria and Fusarium DNA was 84% and 51%, respectively. Mycotoxin alternariol was detected in 100% and emodin in 90% of the samples. The occurrence of deoxynivalenol, diacetoxyscirpenol and T-2/HT-2 mycotoxins was 32%, 42% and 41%, respectively. Plant species had no significant effect on the Alternaria, Cladosporium and Fusarium DNA content of the samples; however, the alternariol, emodin, deoxynivalenol and diacetoxyscirpenol contents differed significantly between plant species. The geographical origin had a statistically significant effect on the Alternaria and Fusarium DNA contents, likely due to differences in weather conditions. Key words: legumes, Alternaria, Cladosporium, Fusarium, quantitative PCR, ELISA Introduction Forage legumes are characterised by high nutritive and protein-balanced values and are the main component of animal feed. They also ensure the preservation and maintenance of soil fertility. The cultivation of legumes is associated with a variety of factors that affect the quality of the final yield. One of these is the effect of fungal diseases, which are often characterised by high harmfulness (Tivoli et al. 1996, Allen and Lenné 1998). According to many researchers, Alternaria Nees, Cladosporium Link and Fusarium Link fungi are the dominant group of mycobiota in forage legumes (Leach 1955, Lager and Johnsson 2002, Al-Askar et al. 2012, Kononenko et al. 2015). Most identified Alternaria and Cladosporium species are characterised as saprotrophic organisms, whereas many Fusarium species although being highly aggressive pathogens for legumes can also lead a saprotrophic life (Leach 1955, Kellock et al. 1978, Zaccardelli et al. 2006, Chittem et al. 2015, Gossen et al. 2016). Analysis of the occurrence of isolated fungi revealed that Alternaria and Cladosporium fungi were the most abun- dant in the mycobiota of alfalfa seeds, with 100% prevalence; the average infection rate of these fungi was 10% and 22%, respectively (Al-Askar et al. 2012). Mycological analysis of the seed samples of four Trifolium species grown in the northwest of the USA found that Cladosporium cladosporioides (Fresen) G.A. de Vries significantly exceeded the occurrence of all other fungal species. This species was present in 11.5% of surface-sterilised seeds and almost 60% of untreated seeds. Alter- naria fungi were the next most abundant (Leach 1955). Alternaria fungi prevailed in the microbiota of field peas in western Canada (Esmaeili Taheri et al. 2017). Fusarium spp. appear to be quite common in Australia in Medicago plants, including, F. acuminatum Ellis et Everh., F. avenaceum (Fr.) Sacc., F. equiseti (Corda) Sacc., F. chlamydosporum Wollenw. & Reinking, and F. graminearum Schwabe (Barbetti et al. 2006). The infection of legume seeds can also be associated with F. incarnatum (Desm.) Sacc., F. verticillioides (Sacc.) Nirenberg and F. solani (Mart.) Sacc. (Lamprecht et al. 1988, Zaccardelli et al. 2006, Al-Askar et al. 2012). Some of these species impede the germination of seedlings and the vigour of germinated seedlings. Fusarium fungi were the most frequently isolated pathogens from the roots of grain legumes in the USA and Canada (Chittem et al. 2015, Gossen et al. 2016). Manuscript received January 2020 A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 266 Forage plants contaminated with fungi often cause various allergies in farmers and animals (Lugauskas et al. 2004, Ajoudanifar et al. 2011, Żukiewicz-Sobczak et al. 2013). Spores of Cladosporium and Alternaria fungi have been shown to be present in significant amounts in the air and can cause severe allergic reactions leading to serious health problems (Hjelmroos 1993, Damialis et al. 2017). Quantitative PCR (qPCR), which reveals the biomass of fungi through the content of their DNA in plant tissues, was used in the current study, has been established as the most convenient method for the quick and objective assessment of infection levels in crops (Nicholson et al. 1998, Pavón et al. 2012, Kulik et al. 2015). This method was successfully applied for the detection and quantifica- tion of the DNA of seven Fusarium species associated with legumes (Zitnick-Anderson et al. 2018, Żelechowski et al. 2019). Some fungal species existent in plant mycobiota have the ability to produce mycotoxins, which have a negative effect on human and animal health (Barbetti and Allen 2005). Used as feed, forage legumes can also be a source of mycotoxins that affect the quality of agricultural products. The species-specific character of mycotoxin production is well known, but it is also affected by environmental factors (Schmidt-Heydt et al. 2009, 2011, Magan and Medina 2016). Previous studies have demonstrated the contamination of forage grasses with Alternaria and Fusarium mycotoxins (Tan et al. 2011, Nichea et al. 2015). An analysis of mycotoxins in legumes in Russia also re- vealed a high occurrence and accumulation of mycotoxin alternariol (AOH) produced by Alternaria fungi, emodin (EMO) associated with Cladosporium fungi, and Fusarium mycotoxins (Gavrilova et al. 2017, Burkin and Konon- enko 2018, Kononenko and Burkin 2018, 2019). The aim of the current study was to assess the presence of Alternaria, Cladosporium and Fusarium fungi, and the mycotoxins they produce in the tissues of Fabaceae plants collected from various regions of European Russia in the context of plant traits. Materials and methods In total, 69 samples belonging to 12 plant species (Galega orientalis, Lathyrus pratensis, Medicago falcata, Med- icago sativa, Melilotus albus, Melilotus officinalis, Trifolium hybridum, T. pratense, T. repens, Vicia cracca, V. sativa and V. sepium) of six genera from the Fabaceae family were collected from different regions of European Russia during the summer of 2015 (Table 1). Manuals were used to determine the plant species, life cycle, growth habit and melliferous capacity (Burmistrov and Nikitina 1990, Gubanov et al. 2003, Skvorcov 2004). Geographically, the analysed samples originated from Leningrad Oblast (n=44), Novgorod Oblast (n=5), Pskov Oblast (n=11) and Smolensk Oblast (n=9). These geographical areas are located throughout the north-western (Leningrad Oblast) and central European regions (Smolensk Oblast). Table 1. Characteristics of the plant species used in this study Genus Species Number of samples Stem growth Life cycle Melliferous capacity (kg ha-1) Galega Tourn. ex L. G. orientalis Lam. fodder galega 4 erect perennial + (60) Lathyrus L. L. pratensis L. meadow vetchling 6 climbing perennial – (30) Medicago L. M. sativa L. alfalfa 4 erect perennial + (170) M. falcata L. sickle medick 2 erect perennial + (170) Melilotus Mill. M. albus Medik. white sweet clover 6 erect annual or biennial + (200) M. officinalis (L.) Pall. yellow sweet clover 3 erect biennial + (200) Trifolium L. T. hybridium L. alsike clover 9 creeping perennial + (145) T. pratense L. red clover 13 creeping perennial + (90) T. repens L. shamrock 7 creeping perennial + (120) Vicia L. V. sepium L. bush vetch 3 climbing perennial – (9) V. cracca L. tufted vetch 10 creeping / climbing perennial + (69) V. sativa L. common vetch 2 erect annual or biennial – (9) A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 267 The plant samples were collected in May and June (n=26), in July (n=20) and in August (n=23). The weather con- ditions are presented in the Table 2. Vegetative plants were cut at a height of 5 cm above the soil surface. The plants were dried at a temperature not higher than 50 °C. The dried plant samples were then separately homogenised in the sterilised grinding chambers of a batch mill (Tube Mill Control, IKA, Königswinter, Germany). The total DNA from 200 mg of milled plant material was isolated using the Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnus, Lithuania) following the manufacturer’s protocol. Using the same kit, DNA was also iso- lated from the mycelium of strains of F. graminearum MFG 58775, A. tenuissima MFP 556081 and Cladosporium sp. MFP 235011 cultivated on potato-sucrose agar. All the fungal strains were maintained in the collection of the Laboratory of Mycology and Phytopathology of the All-Russian Institute of Plant Protection, St. Petersburg, Rus- sia. DNA concentrations from the samples and fungal strains were determined using a Qubit 2.0 fluorometer with a Quant-iT dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and aligned to 2‒50 pg ng−1. The DNA content of Alternaria, Cladosporium and the group of Fusarium fungi producing trichothecene myco- toxins (Tri-Fusarium) were evaluated using qPCR with TaqMan probes (Halstensen et al. 2006, Zeng et al. 2006, Yli-Mattila et al. 2008, Pavon et al. 2012). The reaction was carried out in a 20 μl volume containing 10 μl of a 2 × TaqAB master mix (AlkorBio, St. Petersburg, Russia), 300 nM of each primer, 100 nM of a fluorescent sample (Evrogen, Moscow, Russia) and 2 μl of the DNA sample. All the qPCR assays were run using a thermocycler (CFX 96 Real-Time System; BioRad, Hercules, CA, USA), and the DNA content was presented as the ratio of fungal DNA to total DNA in each sample (pg ng−1). A low quantification limit of 5 × 10−4 pg fungal DNA on one ng of total DNA was established as the DNA threshold value for the samples; this could be quantitatively determined with high precision. All the samples were analysed at least twice. For the mycotoxin detection, 1 g of milled plant material was placed in a tube containing 10 ml of a mix of ace- tonitrile and water (84:16 v/v, respectively). The tube was shaken vigorously and left to incubate for 12–14 h. Af- ter repeated shaking and 10-fold dilution with a buffer solution, the extract was used for an indirect competitive used enzyme-linked immunosorbent assay (ELISA). The determination of T-2/HT-2 toxins, deoxynivalenol (DON), diacetoxyscirpenol (DAS), AOH and EMO was performed using certified kits of immunoreagents and calibrants in- tended for the control of objects of plant origin (VNIIVSGE, Moscow, Russia). The lower limit of the quantitative measurements corresponded to an 85% level of antibody binding. The data were analysed using Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA) and Statistica 10.0 (StatSoft, Tulsa, OK, USA). A standard t-test was used to determine statistically significant differences between the means. Relationships between the quantitative traits were evaluated using the linear Pearson correlation co- efficient (r). The results were considered significant when p < 0.05. Table 2. Weather conditions during 2015 in the sampled regions Region (number of samples) Month Month temperature, °С Average humidity, % Month rainfall, mmmean min max Leningrad Oblast (n=44) May-June 13.8 5.3 25.1 64 68 July 16.9 7.9 25.1 73 86 August 18.2 9.1 28.2 68 47 Novgorod Oblast (n=5) May–June 14.1 3.1 25.4 68 66 July 16.9 7.8 29.7 73 45 August 17.3 5 28.2 71 38 Pskov Oblast (n=11) May–June 16.7 4.4 27.5 93 90 July 18.2 4.2 29.2 72 94 August 19.0 3.8 30.4 71 24 Smolensk Oblast (n=9) May–June 15.9 6.0 27.8 63 62 July 18.8 8.5 31.4 69 61 August 20.1 7.0 32.8 60 7 A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 268 Results The DNA of Alternaria fungi was detected in 84% of the samples. The maximum DNA content of these fungi was found in a Melilotus officinalis sample from Pskov Oblast: 3620 × 10−4 pg ng−1. The average Alternaria DNA content in the analysed samples was 42 × 10−4 pg ng−1. The DNA of Cladosporium fungi was found in 100% of the samples in the range of 7 × 10−4 to 5490 × 10−4 pg ng−1, with an average content of 586 × 10−4 pg ng−1. The maximum Cla- dosporium DNA content was found in a sample of red clover from the Leningrad Oblast. The DNA of Tri-Fusari- um fungi was detected in 51% of the samples in amounts of up to 492 × 10−4 pg ng−1 (i.e. meadow vetchling from Smolensk Oblast). Mycotoxin AOH was detected in 100% of the samples in amounts ranging from 20–1549 ppb, with an average content of 196 ppb. EMO was detected in 90% of the samples in amounts ranging from 17–10000 ppb, with an average content of 652 ppb. DON mycotoxin was detected in 32% of the samples ranging from 100‒631 ppb, DAS in 42% of the samples ranging from 141‒1892 ppb and T-2/HT-2 toxins in 41% of the samples ranging from 4‒27 ppb. The average contents of DON, DAS and T-2/HT-2 toxins were 221, 437 and 10 ppb, respectively. The effect of plant species The occurrence of Alternaria fungi varied widely among the sampled plant species and genera: from 33% in the bush vetch samples to 100% in the samples of common vetch, red clover, shamrock and sickle medick. On average, Melilotus plants accumulated the largest amount of Alternaria DNA compared to plants of other genera (Fig. 1). Plants from the Lathyrus and Trifolium genera were characterised by high levels of Cladosporium DNA compared to other legumes, whereas G. orientalis plants were the least contaminated by these fungi. The occurrence of Tri- Fusarium varied from 25% in the Melilotus officinalis samples to 89% in the Trifolium hybridum samples, but the largest amounts of Tri-Fusarium DNA were detected in Lathyrus plants. On average, the Lathyrus plant samples were the most contaminated by AOH (694±176 ppb), while Vicia and Me- lilotus plants were the least contaminated (53±9 and 91±16 ppb, respectively). On average, samples of Trifolium plants (1284±402 ppb) were the most contaminated by EMO compared to the other legumes (from 19±11 ppb in Melilotus plants to 257±86 ppb in Lathyrus plants). Lathyrus plant samples were most contaminated by Fusarium mycotoxins (DON = 295±99 ppb, DAS = 879 ± 279 ppb and T-2/HT-2 toxins = 11 ± 4 ppb), while none of the Fusar- ium mycotoxins were detected in the Vicia sepium samples. An absence of a significant effect for plant species (12 species) on the amount of Alternaria, Cladosporium and Tri-Fusarium DNA in the samples was established. The effect of the genus (six genera) was substantial only for the amount of Tri-Fusarium DNA. The content of AOH, EMO, DON and DAS mycotoxins in the samples depended on Fig. 1. Alternaria, Cladosporium and Tri-Fusarium DNA contents in samples of legume plants. The bars indicate confidence intervals with a 95% significance level. 0 500 1000 1500 2000 2500 3000 Lathyrus spp. Medicago spp. Melilotus spp. Trifolium spp. Vicia spp. Galega orientalis Co nt en t o f f un ga l D N A × 10 -4 ,p g ng -1 6000 8000 10000 12000 14000 16000 Alternaria Cladosporium Tri-FusariumTri-Fusarium A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 269 the plant species. The effect of plant genus on the accumulation of mycotoxins also proved to be significant. How- ever, the accumulation of T-2/HT-2 toxins was not dependent on either the plant species or genera. The effect of the geographical origin of the plant samples The average amounts of fungal DNA and mycotoxins differed in the legume samples depending on the region of origin (Table 3). The occurrence of Alternaria fungi varied from 56% (Smolensk Oblast) to 93% (Leningrad Oblast), and Cladospori- um fungi was detected in 100% of the examined samples. On average, the samples from Pskov Oblast were most infected by both these fungi. The occurrence of Tri-Fusarium fungi in the regions ranged from 45% (Pskov Oblast) to 60% (Novgorod Oblast), but on average, the samples from Smolensk Oblast were 30–60 times more infected by these fungi. The geographical origins of the plant samples had a significant effect on the amount of Alternaria and Tri-Fusarium DNA, but an effect by this factor on Cladosporium infection was not seen. AOH was found everywhere, whereas the occurrence of EMO in the samples varied from 60% (Novgorod Oblast) to 93% (Leningrad Oblast). DON was not detected in the samples from Novgorod Oblast; the highest frequency of this mycotoxin was found in the samples from Leningrad Oblast and Pskov Oblast (36% each). The occurrence of DAS in the samples ranged from 27% to 48%, and the occurrence of T-2/HT-2 toxins ranged from 18% to 56%. The maximum amounts of AOH, DON, DAS and T-2/HT-2 toxins were detected in the samples from Smolensk Oblast. The samples from Leningrad Oblast were more contaminated by EMO. The geographical origin of the plant sam- ples had a substantial effect on the accumulation of DON and DAS, while the influence of this factor on the accu- mulation of the other mycotoxins was not statistically significant. The effect of the month of sample collection The distribution of samples contamination by fungi and mycotoxins was determined to depend on the month the plants were collected (Table 4). The occurrence of Alternaria fungi increased from 85% in samples collected in May and June to 90% in samples collected in July, but it decreased to 78% in the August samples. Similarly, the occurrence of Tri-Fusarium fungi was 50%, 60% and 52%, respectively. The occurrence of T-2/HT-2 toxins, DON and DAS mycotoxins increased at the end of the growing season. The proportion of samples in which at least one trichothecene mycotoxin was de- tected was 46%, 55% and 70% in May to June, July and August, respectively. The total fungal DNA and all analysed mycotoxin contents in the forage legume samples increased during the growing season. An exception was the content of Alternaria DNA and EMO, which reached maximums in July. The effect of toxins by sampling month on the accumulation of metabolites in the samples was only significant for Al- ternaria and Cladosporium DNA and T-2/HT-2 toxins. Table 3. Fungal DNA and mycotoxin contents in legume plants collected from different regions of Russia in 2015 Geographical origin of plant samples (number of samples) The average amount of fungal DNA ±CI ×10-4, pg ng-1 The average amount of mycotoxins ±CI, ppb Alternaria Cladosporium Tri-Fusarium AOH EMO DON DAS T-2/HT-2 toxins Leningrad Oblast (n=44) 204±43 4306±1356 14±3 182±26 836±279 63±14 152±26 5±1 Novgorod Oblast (n=5) 176±65 2707±445 33±22 58±12 55±28 0 60±36 2±1 Pskov Oblast (n=11) 857±387 10572±4741 17±13 192±79 99±27 55±25 94±47 1.4±0.7 Smolensk Oblast (n=9) 62±33 9466±3191 965±567 343±162 252±73 192±95 564±267 9±3 CI = confidence interval with significance level of 95% A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 270 The effect of plant habit Sampled plants with erect stems (V. sativa, Melilotus spp., G. orientalis and Medicago spp.) had a lower occur- rence of Alternaria (71%) and Tri-Fusarium (42%) fungi than the samples of plants with creeping stems (Lathyrus spp., Trifolium spp., V. sepium and V. cracca): 91% and 60%, respectively. On average, the Alternaria and Tri-Fusarium DNA contents did not differ significantly for plant samples with erect or creeping stems (Fig. 2). However, a significant effect of stem growth on infection with Cladosporium fungi was revealed. On average, plants with creeping stems accumulated the DNA of these fungi 4.5 times more than plants with erect stems. The occurrence of trichothecene mycotoxins in the erect-stem plant samples was 2.5–5 times lower than in the creeping-stem plant samples. The proportions of plant samples with erect and creeping stems, in which at least one trichothecene mycotoxin was found, were 28% and 70%, respectively. Plants with erect stems were signifi- cantly less contaminated by all the analysed mycotoxins. The most substantial difference was for EMO; its content in creeping plants was 17 times higher than in erect plants. The AOH and trichothecene mycotoxin contamination of the creeping plants was 2.5–6 times more than that of the erect plants. Plant habit had a significant effect on the accumulation of all the analysed mycotoxins. The effects of life cycle and melliferous capacity The occurrence of Alternaria fungi in the annual/biennial legume (Melilotus spp. and V. sativa) and perennial leg- ume plant samples (G. orientalis, Lathyrus spp., Medicago spp., Trifolium spp., V. sepium and V. cracca) was 73% and 88%, respectively. At the same time, the perennial legume samples were 2 times less contaminated with Al- ternaria fungi compared to the annual/biennial plants. Cladosporium fungi were found in 100% of the samples, but no significant effect of life cycle on the accumulation of Cladosporium DNA was found. The occurrence of Tri- Fusarium fungi did not depend on life cycle either; they were detected in half of the samples in each group: 55% and 53%, respectively. However, perennial legume plants contained an average of 10 times more Tri-Fusarium DNA than the annual/biennial plants (Fig. 3A). Table 4. Alternaria, Cladosporium and Tri-Fusarium DNA and mycotoxins in samples of legume plants collected at different times during the 2015 growing period The month (number of samples) The average amount of fungal DNA ±CI ×10-4, pg ng-1 The average amount of mycotoxins ±CI, ppb Alternaria Cladosporium Tri-Fusarium AOH EMO DON DAS T-2/HT-2 toxins May-June (n=26) 164±51 285±54 18±6 156±28 580±254 53±15 111±30 1.5±0.5 July (n=20) 585±224 8133±2680 17±7 199±52 118±24 40±15 136±41 3±1 August (n=23) 169±46 10192±2714 385±235 239±71 999±448 126±42 325±111 8±2 CI = confidence interval with significance level of 95% 0 200 400 600 800 1000 1200 0 2000 4000 6000 8000 10000 12000 Alternaria Cladosporium Tri-Fusarium AOH EMO DON DAS Т-2 Co nt en t o f m yc ot ox in s, p pb Co nt en t o f f un ga l D N A ×1 0- 4 , pg n g- 1 erect stem creeping stem Tri-Fusarium DNA mycotoxins AOH EMO DON DAS T-2/HT-2 Fig. 2. Fungal DNA and mycotoxins contents in legume plant samples with different stem growths. The bars indicate confidence intervals with a 95% significance level. A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 271 The occurrence of all analysed mycotoxins in the annual/biennial plant samples was 2–6 times less than in the perennial grasses. The perennial legumes also contained more of all the analysed mycotoxins compared to the annual/biennial plants, but this difference was not statistically significant for any of the mycotoxins. On average, the occurrence of Alternaria and Tri-Fusarium fungi in melliferous legume plants was higher (90% and 57%, respectively) than in non-melliferous legumes (64% and 36%, respectively), while Cladosporium fungi were found in 100% of the samples. The contamination of melliferous and non-melliferous legumes with Alternaria and Cladosporium fungi was not significantly different (Fig. 3B). Non-melliferous plants were 12 times more contaminated with Tri-Fusarium fungi than melliferous plants, and this difference was significant. The occurrence of AOH and EMO mycotoxins in the samples of melliferous and non-melliferous legumes was ap- proximately the same, ranging from 88% to 100%. The occurrence of trichothecene mycotoxins in the non-mel- liferous legumes was in the range of 28%–40%, which was less than the similar values for melliferous plants, in which the occurrence of these mycotoxins was 55%‒64%. The contamination of non-melliferous legumes by AOH, DON and DAS trichothecene mycotoxins was 1.5–5 times higher than for melliferous plants and the difference was significant. The opposite tendency was observed for EMO: this toxin accumulated in non-melliferous legumes 4.5 times less than in melliferous ones, but it was not statistically significant. Discussion Differences in the habits and life cycles of legumes complicate the correct analysis of their microbiological contami- nation. The facility of molecular methods, whereby DNA is extracted in the first stage from an average homogene- ous sample obtained by grinding whole plants, represents great advantages compared with traditional methods. The active application of molecular methods to assess the microbiological status of plants has been observed in recent years (Kulik et al. 2015, Gagkaeva et al. 2017, Zitnick-Anderson et al. 2018). In the mycobiota of forage legumes, Alternaria, Cladosporium and Fusarium fungi have been found to be the dominant group (Leach 1955, Lager and Johnsson 2002, Al-Askar et al. 2012, Kononenko et al. 2015). In our study, Cladosporium fungi were also the most common among the three analysed groups of fungi, and they were found in all the samples. The most favourable substrates for these fungi were Lathyrus and Trifolium plants, in which a high content of Cladosporium DNA was found. The DNA content of these fungi in the group of plants with creep- ing stems was significantly higher than in plants with erect stems. According to Tivoli et al. (1996), as a result of higher humidity at the base of the pea canopy, greater development of leaf blight is observed in the lower part of pea plants. In support of this, accessions of Trifolium pratense with erect growth habit were less susceptible to rot compared with creeping forms of red clover (Vleugels et al. 2013). 0 2000 4000 6000 8000 10000 annual/biennual perennial Co nt en t o f f un ga l D N A × 10 -4 , p g ng -1 Alternaria Cladosporium Tri-FusariumTri-Fusarium Fig. 3. Fungal DNA content in legume plant samples with different (A) life cycles and (B) melliferous capacity. The bars indicate confidence intervals with a 95% significance level. 0 2000 4000 6000 8000 10000 melliferous non-melliferous Co nt en t o f f un ga l D N A × 10 -4 , p g ng -1 BA A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 272 In the spring samples, the content of Cladosporium DNA was significantly lower compared with the plants col- lected later in the year. The colonisation of pea (Pisum sativum) leaves by fungi has been previously described; an increase of predominant Cladosporium spp. by the end of the growing season has been observed (Dix and Web- ster 1995). Probably, the dramatic prevalence of Cladosporium conidia in the air compared to the number of other fungal conidia (Grinn-Gofroń et al. 2016) is associated with the significant contamination of plants by these fungi. On average, the highest amount of Alternaria DNA was found in the Melilotus plants. The geographical origin and the month sampled had a statistically significant effect on the content of Alternaria DNA, while the effects of plant species, genus and traits were accidental. Therefore, environmental conditions had the greatest effect on the distribution of these fungi. The contamination of legume plants by Alternaria fungi reached a maximum in July compared with samples collected earlier and later. According to available weather conditions in the ana- lysed regions, July 2015 was characterised by the highest rainfall in most areas. The correlation between rainfall and Alternaria DNA content was significant (r=+0.28), while the average temperature and humidity were not sub- stantial. Differences in the Alternaria DNA content between the regions were congruent: samples from the Pskov Oblast with the highest rainfall were the most infected, while samples from the Smolensk Oblast with the mini- mum rainfall were the least infected. It is known that the penetration and growth of fungi in plant tissues are af- fected by environmental conditions, but according to some researchers, the effect of rainfall during the growing season has a more significant effect on the development of Alternaria leaf blight than temperature (Saharan and Saharan 2004, Meno et al. 2019). Tri-Fusarium fungi producing trichothecene mycotoxins were found in half of the analysed legume samples. On average, a significantly higher content of their DNA was found in the Lathyrus plants. The geographical origin of the samples had a statistically significant effect on the content of Tri-Fusarium DNA, while the effects of sampling month, plant species, genus and traits were accidental. In the plant samples from the Smolensk Oblast, the aver- age content of Tri-Fusarium fungi was higher than in samples from other regions. The weather conditions in this region were characterised by the highest average temperature for all the summer months during 2015 compared with the other analysed regions. The correlation between the average monthly temperature and the Tri-Fusarium DNA content was significant (r=+0.29). Analysis of the relationships between the analysed fungi revealed a significant positive correlation between the Alternaria and Cladosporium fungi DNA contents in the plant samples (r=+0.37). In the samples collected in July and August, this connection was more substantial (r=+0.46 and +0.56, respectively). When analysing the fungi contamination of samples grouped according to traits, this connection was even more significant: in plants with creeping stems, the correlation coefficient between the Alternaria and Cladosporium fungi DNA contents was r=+0.47, and in the annual/biennial plant samples, its value reached r=+0.50. This demonstrates the similarity of the conditions required for the growth of Alternaria and Cladosporium fungi, especially in conditions favourable for saprotrophic fungi (i.e. proximity of the soil and aging plant tissue). Previous molecular analyses have revealed a similarly significant relationship between Alternaria and Cladosporium fungi coexisting in small grain cereals (Kulik et al. 2015, Gagkaeva et al. 2017) and legumes (Orina et al. 2018). Our study did not reveal any significant effect of sampling month on the accumulation of mycotoxins in legumes tissues. A tendency for mycotoxin accumulation over the growing season was not found in T. pratense, Medicago spp. or Melilotus spp. plants collected from other regions of Russia (Burkin and Kononenko 2018, Kononenko and Burkin 2018). In addition, there were no differences in the content of Fusarium mycotoxins in naturally growing herbs from the Poaceae family collected in spring and summer in one region of Argentina (Nichea et al. 2015). However, the EMO, AOH, DON and DAS contents in samples of Lathyrus pratensis collected in August were signifi- cantly higher than in plant samples collected in May and June (Kononenko and Burkin 2019). No statistically significant effect of life cycle on the amount of fungal DNA or mycotoxin content was detected, although, according to our results, the perennial legumes contained more of all the analysed mycotoxins compared with the annual/biennial plants. This is consistent with Engels and Krämer (1996), who found high T-2 toxin, ZEA and, especially, DAS content in perennial ryegrass compared with annual ryegrass. According to our results, the content of Alternaria and Cladosporium DNA in melliferous and non-melliferous legumes was not significantly different. The opposite situation was found in the case of the Tri-Fusarium fungi: non-melliferous legumes were contaminated with significantly higher amounts of Tri-Fusarium DNA than mel- liferous ones, as well as with DON and DAS mycotoxins. According to some researchers, flowering plant pollen can stimulate conidia germination and the growth of fungi on leaf surfaces by reducing nutritional competition. A G R I C U LT U R A L A N D F O O D S C I E N C E A.S. Orina et al. (2020) 29: 265–275 273 The abundance of pollen in the atmosphere coincides with an increase in the numbers of Alternaria and Clad- osporium conidia during the summer months, and pollen and spores are deposited together on plant surfaces in great numbers (Dix and Webster 1995). Bees can also contribute to plant infection by transferring fungal conidia. Analyses of bee pollen have revealed the ubiquitous presence and predominance of Alternaria, Mucor, Aspergil- lus and Cladosporium fungi, and Aureobasidium and Penicillium have also often been found, whereas Fusarium fungi have not been detected (Osintseva and Chekryga 2008, Kačániová et al. 2011, Deveza et al. 2015). A correlation between the content of Alternaria DNA and mycotoxin AOH in all the analysed legume samples was not detectable. This was probably because the current study analysed the presence of these fungi at the genus level, while different Alternaria species are characterised by a contrasting ability to produce this mycotoxin (An- dersen et al. 2002). Additionally, the correlation between the Cladosporium DNA and mycotoxin EMO contents was significant (r=+0.38). Various fungi can produce EMO (Kusari et al. 2008), and this is also a known metabolite of some plants (Izhaki 2002). Cladosporium fungi are probably not the exclusive producers of this mycotoxin in legume tissues; however, the contribution of these fungi is substantial. A correlation was established between the Tri-Fusarium DNA and DON and DAS mycotoxins contents (r=+0.39 for each) observed in the group of creeping- stem plants (r=+0.31 and +0.33, respectively) and perennial plant samples (r=+0.34 and +0.36, respectively). The correlation between the trichothecene mycotoxin contents was also significant (T-2/HT-2 toxins and DON=+0.48, T-2/HT-2 toxins and DAS=+0.52, and DON and DAS=+0.85). The current study’s findings on the distribution of fungi and their relationships with host plants and each other are of high scientific and practical interest. Knowledge about the contamination of legumes with fungi and mycotoxins is important for human and animal health. 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