Corynespora cassiicola IN SOYBEAN SEEDS – INCIDENCE AND TRANSMISSION Corynespora cassiicola EM SEMENTES DE SOJA: INCIDÊNCIA E TRANSMISSÃO

The fungus Corynespora cassiicola, the causal agent of the target spot in soybeans, has been considered, from the seed pathology point of view, a seed-borne pathogen of limited importance. Therefore, little importance has been given to the role of the seeds in the transmission of this pathogen. The objectives of this study were to determine the incidence of C. cassiicola in soybean seeds and evaluate the effects of this seed-borne pathogen, inoculated in the seeds, concerning to physiological and epidemiological parameters. The experiments were carried out at TAGRO and Embrapa Western Agriculture under lab (blotter test and seed germination test) and greenhouse conditions (growing on test). The fungus C. cassiicola was detected in 11.3% of the 639 seed samples analyzed, with an average incidence of 0.91% and a maximum of 8.5%. The transmission of C. cassiicola from the seeds to above-ground parts of soybean seedlings was demonstrated, by pathogen establishment on the cotyledon, showing circular lesions with concentric rings, reddish-brown in the center and surrounded by a yellowish-green halo, as a typical symptom of target spot. Reddish-brown lesions on the roots and stem of the seedlings were also observed. Considering a sample seed with 66.0% of C. cassiicola incidence, the symptomatic transmission based on cotyledon symptoms was 42.2%, corresponding to a transmission rate of 2.4:1. This is the first report, in a quantified way, about the transmission of C. cassiicola from the seeds to above-ground parts of soybean seedlings. When compared to non-inoculated seeds, seed germination, seedling emergence, and seedling initial development were influenced by the presence of the pathogen in the seeds, with the lowest values being observed when the seeds were inoculated.


INTRODUCTION
Target spot of soybean (Glycine max (L.) Merr.) caused by Corynespora cassiicola (Berk. & Curt.), was first detected in Brazilian soybean fields in 1976, in São Paulo State 1976). Ten years later, the disease was reported in the States of Mato Grosso, Mato Grosso do Sul and Rio Grande do Sul (YORINORI, 1989). According to Godoy (2015), the incidence of this disease has increased in the last crop seasons due to the lower sensitivity/resistance of the fungus to the fungicides most commonly used in soybean crops and/or the introduction of more susceptible host genotypes. Yield losses of 18 to 32% have been reported on susceptible cultivars 2015). This pathogen spread mainly by wind and spattering of rain 2005) and information about the transmission and spread through soybean seeds was not found.
The fungus C. cassiicola has been considered, from the seed pathology point of view, a seed-borne pathogen of limited importance. Therefore, little importance has been given to the role of the seeds in the transmission of this pathogen.
The incidence of this pathogen in the seeds is unusual. However, it can be detected at low levels. In this context, Roim et al. (1999) detected the presence of C. cassiicola in soybean seeds, with an average incidence ranging from 0.2% to 1.3%. Seeds naturally infected, hardly will show high levels of C. cassiicola. However, results obtained by Camargo et al. (2013), showed an average incidence of this pathogen in the seeds ranging from 18.0% to 43.0%. According to these authors, this can be attributed to the occurrence of favorable environmental conditions for the target spot, particularly rainfall at harvest. This is an uncommon result, being an exception to the rule.
Only a few references were found about the possibility of pathogen spread through infected seeds (SINCLAIR; BACKMAN, 1989;GODOY et al. 2015). There is no guarantee that seed-associated pathogens will infect seedlings, so the simple incidence of pathogens in seeds does not implicate their transmissibility. No scientific studies related to the transmission of C. cassiicola by soybean seeds were found. Consequently, from the epidemiological point of view, the role of soybean seeds as a source of primary inoculum of the target spot is still unknown. Due to this, transmission efficiency from seed to seedling should be demonstrated and quantified.
The objectives of this study were to determine the incidence of C. cassiicola in soybean seeds produced in different Brazilian locations and evaluate the effects of this seed-borne pathogen concerning to physiological (seed germination, seedling emergence, and initial development of soybean seedlings) and epidemiological parameters (transmission of C. cassiicola by seeds to aboveground parts of soybean seedlings).
To determine the incidence of C. cassiicola in soybean seeds, 639 seed samples from different cultivars and produced in different Brazilian locations (Goiás, Mato Grosso, Mato Grosso do Sul, Paraná, Santa Catarina e São Paulo States) were analyzed, using the blotter test method. This work was carried out in TAGRO -Tecnologia Agropecuária Ltda, Londrina, PR. The Blotter test was performed according to international recommendations (NEERGAARD, 1979), with some modifications (GOULART, 1984). Four hundred soybean seeds per sample were placed into transparent plastic boxes -Gerbox® type -(20 seeds per gerbox), containing three sheets of qualitative filter paper previously moistened in diluted agar (10 g of agar/1000 ml of water) and incubated under 22 °C with alternating 12 h period of darkness and fluorescent white light, where they remained for seven days. After this period, the presence of C. cassiicola in the soybean seeds was recorded using a stereoscopic microscope. The pathogen identification was based on their morphological characteristics. The results were expressed in the percentage of contaminated seeds.
The effects of this seed-borne pathogen on physiological and epidemiological parameters were evaluated in experiments developed at Embrapa Western Agriculture, Dourados, MS, in October 2015, using soybean seeds inoculated with C. cassiicola (66% of incidence). Seeds were inoculated according to the methodology developed by Tanaka, Menten, and Marianno (1989). Before inoculation, seeds asepsis was made by immersing them in sodium hypochlorite solution (1%) for 3 min. Pure culture of C. cassiicola developed in PDA medium was used as inoculum. Plates were incubated in an incubation chamber under 22°C with alternating 12 h period of darkness and fluorescent white light, where they remained for 7 days. In each plate 50 seeds were placed in contact with the surface of the colonies, which was manually shaken for 30 seconds, allowing the adhesion of the inoculum to the surface of the seeds. The plates containing the inoculated seeds remained for 48 hours under laboratory conditions and then the seeds were air-dried for 24 hours on absorbent paper.
The seed germination was performed with one subsample of 50 seeds per replication, in a total of 200 seeds per treatment. The seeds were distributed on two sheets of Germitest® type paper rolls, moistened with distilled water in the proportion of 2.5 times the substrate dry mass. Next, the paper rolls were placed inside the germinator at 25 ºC for 8 days. After that period, the normal seedlings were counted, and the results were expressed in percentage of normal and abnormal seedlings (BRASIL, 2009).
The physiological and epidemiological parameters were evaluated using the "growing on test" in experiments carried out in greenhouse conditions.
Seeds were sown in plastic trays (56x35x10cm), containing sand previously sterilized. Two hundred seeds for each treatment were planted by placing them in equidistant 3-cmdeep wells. The moisture was maintained through irrigation, according to the needs of the seed. The final evaluation was done 15 days after sowing computing the percentage of emerged normal seedlings, height and fresh weight of shoot, and root of soybean seedlings.
The occurrence of lesioned seedlings by the pathogen at 15 days after sowing was evaluated. These injured seedlings were collected and taken to the laboratory for analysis. Fragments of cotyledons and hypocotyl/root from these seedlings were washed and disinfested with 70% alcohol and 2% sodium hypochlorite, and then dried on sterile filter paper. After that, the fragments were incubated in humid chamber under 22 °C with alternating 12 h period of darkness and fluorescent white light. Five days after incubation, the fragments were evaluated under stereoscopic microscope and fungal colonies were observed. The data obtained in this evaluation were used to calculate the transmission and rate of transmission of pathogen from the seeds to aboveground parts of soybean seedlings.
The data were submitted to variance analysis and the Student's t test was used to compare the means of the treatments.
The results about the occurrence of C. cassiicola in soybean seeds are shown in Table 1.
After the incubation period in the seed health test, it was observed, on the seeds, colonies ranging in color from gray to dark gray, with the presence of dense aerial mycelium ( Figure 1A; 1B), conidiophores and conidia ( Figure 1C), typical of C. cassiicola, which was confirmed on the basis of their morphological characteristics, according to Barnett;Hunter (1972).
In 2008, an analysis of the sanitary quality of a seed sample from a commercial soybean crop, cultivar, FT Jaciara, susceptible to the target spot, from Campo Novo do Parecis, MT, revealed 27.0% of C. cassiicola incidence. This result, although rare, suggests the possibility of the occurrence of this pathogen in the seeds at high levels, corroborating with those achieved subsequently by Camargo et al. (2013).
In the 639 analyzed samples, C. cassiicola was detected in 11.3% of them (frequency). The pathogen was recorded in every year the work was done, especially in 2011, with 23.8% of the samples showing the pathogen, when compared to the results obtained in the other years. It should be emphasized that this large number of positive seed samples with C. cassiicola in that year, did not reflect in high incidence of the pathogen in seeds (average incidence = 0.38% and maximum incidence = 2.25%). In general, the average incidence of this pathogen in the seeds was 0.91%, with the highest incidence (8.5%) being observed in the seeds produced in 2015. The data obtained in the present study also corroborate with other researches that demonstrated the low levels of this pathogen in soybean seeds (ROIM et al, 1999).
From the year 2013 onwards, there was a trend in the increase of the occurrence of this pathogen in seeds, in terms of frequency and incidence, according to data presented in Figure 2. Considering that the incidence of this pathogen in the seeds is unusual, these results suggest that the presence of this pathogen in the seeds, to a greater or lesser incidence, can probably be attributed to the increase in incidence and severity of the target spot in soybean crops. According to Godoy (2015) reports, emerging target spot epidemics can be explained by either or both the lower sensitivity/resistance of the fungus to the fungicides most commonly used in soybean crops and introduction of more susceptible host genotypes.
Detection of phytopathogens in seeds has become increasingly important especially because of the relevance of this measure as part of plant health management (SOUSA; SIQUEIRA; MACHADO, 2016;SAJEESH et al.;2014).
The seed health testing (blotter test) used in this research for the detection of C. cassiicola in soybean seeds was adequate for this purpose.
The results regarding the relation of this seed-borne pathogen with physiological and epidemiological parameters, using inoculated seeds with C. cassiicola (66.0% de incidence), are shown in Table 2.
The transmission of C. cassiicola from the seeds to above-ground parts of soybean seedlings was demonstrated, by pathogen establishment on the cotyledon, shortly after the emergence, showing circular lesions with concentric rings, reddish-brown in the center and surrounded by a yellowish green halo, as a typical symptoms of target spot (Figura 1D). According to Lamotte et al., (2007), this yellowish color surrounding the lesions is a consequence of the toxin cassiicolin action, produced by pathogen, which is released into the cells and kills the tissues adjacent to the site of infection.
Considering a seed sample with 66.0% of C. cassiicola incidence, the symptomatic transmission of this fungus, based on the cotyledon symptoms, was 42.2%, corresponding to a transmission rate of 2.4:1 (Table 2). It is known that the transmission process of a particular pathogen varies according to its incidence on the seeds. It means that the higher pathogen incidence on the seeds, the higher percentage of transmission. Based on these results, it can be inferred that infected seeds with C. cassiicola can play an important epidemiological role as source of primary inoculum of the disease in the field.
Seed germination, seedling emergence and seedling initial development (height and fresh weight of shoot and root) were influenced by the presence of the pathogen in the seeds, with the lowest values being observed when the seeds were inoculated (Table 2). Seedlings originated from inoculated seeds showed reduction of initial development (lower height), compared to those from non-inoculated seeds, as can be seen in Figure  1E. Reddish-brown lesions, superficial and without constriction, were also observed on the roots and stem of the seedlings, due to the presence of this pathogen in seeds ( Figure 1F). Infected tissues were collected and taken to the laboratory for analysis. After the incubation period, fungal colonies were observed. Based on morphological characteristics, the pathogen was identified as C. cassiicola ( Figure  1G). This is the first report, in a quantified way, of the transmission of C. cassiicola from the seeds to above-ground parts of soybean seedlings. It is important to state here that these results were obtained with artificially inoculated seeds, since the natural incidences are very lower than that used in the experiment. The present research provides information for pathology and for production of soybean seeds, since, nowadays, there are no studies determining this transmission.