Sanity, Pathogenicity, and Transmission of Fungi associated with Seeds of Chia coming from the Southern Region of Tocantins

— A seed of Chia ( Salvia hispânica L.) has been growing, assuming significant values of consumption in the current society, due to its high nutritional power. However, its seeds can be targeted by the presence of fungi. The aim of this study was to evaluate the health, pathogenicity, and transmission of fungi associated with seeds in Chia culture in the southern Tocantins. For the work, Chia seeds were collected from the UFT experimental unit, Gurupi-TO, produced from the combination of five levels of phosphate fertilization (0, 30, 60, 90 and 120 kg ha -1 ) and five levels of nitrogen fertilization, 30, 60, 90 and 120 kg ha -1 ). The filter paper method ( blotter test ) was used to perform the mycobacterial survey. The pathogenicity and transmissibility of fungi associated with seeds were also tested. The genus detected in the superficial part of the seeds were: Cladosporium sp., Phoma sp., Fusarium sp., Alternaria sp. and Curvularia sp. The Chia seeds of the municipality of Gurupi-TO presented low incidence of fungi. There was no pathogenicity and transmissibility of the fungi detected in Chia seeds in the seedlings.

scarce due to the fact that it is a poorly explored culture. However, there are reports of diseases caused by fungi of economic importance that have affected the crop in the last harvests [7][8][9]. The presence of fungi in seeds, besides allowing the introduction of pathogens in areas of cultivation and causing diseases to agricultural crops, can also cause alterations in the metabolism of plants compromising their therapeutic properties and flavor. Several species of fungi cause physicochemical alterations in the tissues of the seeds, causing loss of lipids, carbohydrates, proteins, and fatty acid increase, besides influencing seed germination [10,11]. The detection of occurrence, associated pathogenicity, and transmission of fungi in seeds can facilitate the choice of disease control measures, enabling the elaboration of identification and control methodologies. However, there are few related studies on the presence of fungi in Chia seeds produced in Brazil. For this reason, the present work had as objective: to evaluate the sanity, pathogenicity, and transmission of fungi associated with seeds in Chia culture produced in the southern region of Tocantins.

II. MATERIAL AND METHODS
The experiment was carried out at the Laboratory of Phytopathology and greenhouse of Universidade Federal do Tocantins (UFT), Gurupi-TO.
The tests were made approaching 1 -Sanity, 2 -Pathogenicity, 3 -Transmission of fungi associated with seeds. Therefore, the seeds were collected in the experimental field of the UFT located at the coordinates with 11 • 43' 45"S and 49 • 15´W average altitude of 285 m. The climate of the region is of the mega thermic type with summer rains and dry winter [12]. For the tests, 25 samples of Chia seeds produced from the combination of five levels of phosphate fertilization (0, 30, 60, 90 and 120 kg ha −1 ) and five levels of nitrogen fertilization (0, 30, 60, 90 and 120 kg ha −1 ) were used in the crop 2015/2016 in the municipality of Gurupi-TO.

Test 1 -Sanity 1
For this assay, we used the filter paper method (blotter test) according to the Manual of Sanitary Analysis of Seeds (Brazil, 2009) [13]. The experimental design was completely randomized (CRD) with 4 replications, each one in a Petri dish containing 50 seeds of each treatment. The Petri dishes containing the seeds were placed in the freezer for 12 hours to inhibit the germination. After this period, the Petri dishes were transferred to incubation chamber at 25 ± 2•C and photoperiod of 12 hours for 7 days. At the end of this period, the fungi were surveyed from the individual analysis of the seeds with the aid of the stereoscopic optical microscope visualizing the morphological characteristics of the fungal structures, the fungi were identified as Based on specialized literature such as Ellis (1971) [14], Barnett & Hunter (1998) [15] and Watanabe (2010) [16]. The fungi found were isolated and cultivated in potato-dextrose-agar culture medium (PDA) for the pathogenicity assay. The fungi incidence data were expressed as percentages (%).

Test 2 -Pathogenicity
The pathogenicity of the fungi transported by the seeds was evaluated by inoculations in the aerial part of seedlings. To obtain the seedlings, Chia seeds were sown in pots of 8 dm3 containing as substrate the mixture of soil and sand autoclaved at 120 • C for 1 hour, in the proportion 2:1. Each vase with 4 seeds, at 45 days after sowing, with the aid of a manual spray was made the inoculation of the suspension 106 spores/ml resulting from each fungal isolate (Cladosporium sp., Phoma sp., Fusarium sp., Curvularia sp. and Alternaria sp.), adjusted with Neubauer chamber. The suspensions were prepared from pure colonies of fungi cultivated in culture medium (PDA) incubated 7 days, under a temperature of 25 ± 2 • C and photoperiod of 12 hours. Similarly, disc inoculations were made on the stems of the plants. Using as a witness a plant sprayed only with sterile water. The inoculated seedlings were packed in a moist and dark chamber for 48 hours. After this period, they were transferred to a greenhouse, where they remained for 10 days. The evaluations of the symptoms were performed ten days after inoculation. Subsequently, indirect isolation of the fungus was performed, according to Alfenas & Mafia (2007) [17] with the purpose of confirming the causal agent, fulfilling Koch postulates. The experiment was assembled according to a completely randomized design (CRD) with 6 treatments (5 fungi + control), and 4 replications. Each repetition consisted of a plant.

Test 3 -Transmission of seed-associated fungi.
For the transmission test, 50 seeds of each treatment were sown in styrofoam trays. Previously disinfected with 1% sodium hypochlorite, one seed per cell, containing Nutrimax commercial substrate, autoclaved at 120 • C for 1 hour. After sowing, the trays were transferred in a greenhouse and irrigated daily. The seedling evaluation was performed at 5, 10, 15, 20, 25 and 30 days. After germination, none of the seedlings presented symptoms characteristic of the diseases, so the Koch postulated was not made, which is a requirement solely for confirmation of the existing pathogen.

III. RESULT AND DISCUSSION
In the sanity test, a total of five fungi genera (Cladosporium sp., Phoma sp., Fusarium sp., Alternaria sp., and Curvularia sp.) associated with Chia seeds were detected. The results of fungal incidence are presented as percentages in Table1.  [18] when studying the same species Salvia hispânica L. also determined the presence of contaminating filamentous fungi in seven commercial samples of seeds evaluated observing the presence of the following fungi genera: Aspergillus sp., Aspergillus niger., Aspergillus flavus, Penicillium sp., Cladosporium and Curvularia sp. In Brazil, the genera of fungi that most frequently attack the seeds of medicinal, condiment and aromatic plants, highlight the Aspergillus, Epicoccum, Mucor, Penicillium, Rhizopus and Trichoderma considered contaminants in seeds, Cladosporium, Curvularia and Pestalotiopsis, considered contaminants also however in some cases can cause diseases, Bipolaris, Alternaria, Fusarium, and Phoma, which have species that can cause important diseases, as the host involved [19]. For this essay, it was observed that in the Chia seed samples, from high and low Nitrogen (N) and Phosphorus (P) doses, the genus Cladosporium sp. presented the highest percentages of fungi. The fact that it was detected in 100% of the samples analyzed and the incidence of this pathogen in the seeds ranged from 5% to 36%. Observing

International Journal of Advanced Engineering Research and Science (IJAERS)
[ Vol -6, Issue-8, Aug-2019]  https://dx.doi.org/10.22161/ijaers.68.26  ISSN: 2349-6495(P) | 2456-1908(O) the highest incidence in the 90 doses of N and 120 P (Table 1). Reis et al., (2007) [20], when evaluating six lots of commercial seeds belonging to the family of Lamiaceae, also evidenced the presence of Cladosporium sp., reaching more than 20% of incidence. Most fungi of the genus Cladosporium sp. are cosmopolitan and their distribution is commonly found in plants and seeds [21,22]. Depending on the fungal species and host plant, there may be a lower or higher incidence and may cause important lesions or death in plants. High incidence levels of Cladosporium sp. are common in seeds from the field or during storage, causing damage to germination and vigor, especially when the treatment is not done in seeds [23,24]. According to Braga et al., (2010) [25], the high or low incidence of Cladosporium sp may also be influenced by the drying period, environment conditions or storage period. It is important to highlight the fact that few are the studies addressing the occurrence of Cladosporium sp. in Chia seeds.
The second most frequent genus in the seeds evaluated was Fusarium sp and the seeds were from the 60-90 dose of N and P that presented the highest incidence (9%). This fungus is constantly associated with seeds of different plant species and its aggressive and rapid growth can cause the death of Seeds [26]. The Fusarium is considered pathogenic, because it causes reduction of the germinative capacity and diseases in the plant, this fungus presents a wide range of host and high economic expression, besides being producing mycotoxins [27,28]. There are reports of the genus Fusarium sp. causing diseases in aromatic and medicinal plants (Mentha piperita L.) compromising different parts of the plants [29]. Several species of this fungus are sources of soil inoculum, causing problems such as seed rot, root necrosis, rot, mold, biochemical seed transformation, widespread wilting and seedling death [10,30]. Other important genera found in the sanity test were Alternaria sp. and Curvularia sp. presenting similar percentages in the samples evaluated, with variation between the samples of 1-5% of incidence. Kruppa Goulart (2004) [27], the genus Alternaria sp. is very common in seeds, being, in most cases, associated only as a saprophyte and depending on the incidence can cause impairment in seed quality. However several species of Alternaria sp. and Curvularia sp. are associated with diseases in many plant species [19,32]. According to Machado (1988) [33], The pathogen maintains its viability and characteristics for a long time, allowing its dissemination in new areas quickly and with a high probability of causing disease after planting. For the genus Phoma sp. In the sanity test, we observed a low percentage occurrence of these fungi in Chia seeds produced in high and low doses of N and P. There was an occurrence less than or equal to 1% among the samples evaluated. Different species of cultivated and wild plants have shown the similarity of fungi of the genus Phoma sp. [34]. This fungus has caused diseases in several species of plants, there are reports of species of Phoma sp. about symptoms in the stems and leaf in adult specimens of this host [35]. Machowicz-Stefaniak et al., (2008) and Zimowska (2008) [36,37], evaluating the presence of Phoma exigua in sage, thyme, and lemon balm plants, observed inhibition in germination and necrotic stains on the stem and leaves. According to all the work reported above, the percentage of fungi found in Chia culture is low compared to other Lamiaceae families, where the highest incidence of this genus was observed. It was also observed that the control presented percentages not equidistant or equal to the higher doses than seeds produced in low or high doses of nitrogen and phosphorus did not interfere in the percentage of fungi observed. Justifying that the percentages of fungi present in the evaluated samples may be due to favorable climate conditions in their development, such as precipitation during the maturation and harvest periods that often occur in the region, or to successive cultivations of the same species or species belonging to the same family, in the same production field or due to storage. This is why the need for better management of productive fields, harvesting, and storage techniques is evident, in order to reduce the incidence of fungi and thus ensure a better sanitary quality of the seeds in Chia crop.

Pathogenicity of fungi associated with Chia seeds.
Regardless of the part of the plant in which the fungi Cladosporium sp., Phoma sp., Fusarium sp., Alternaria sp. and Curvularia sp. were inoculated, no pathogenic action was observed (Table 2).

International Journal of Advanced Engineering Research and Science (IJAERS)
[  Reports from several studies confirm that the presence of microorganisms, even pathogenic, in the seed, is not sufficient to ensure that it will infect the plant originating from this seed, because, there are several factors that can limit the efficiency of transmission of pathogens present in seeds such as -inoculum quality, soil physical factors, temperature, humidity, oxygen, survival time of the pathogen in the seed [33,41]. What may have occurred with the pathogenic agents present in Chia seeds, which were not transmitted to seedlings due to lack of favorable conditions, however, the seed-pathogen and possible relationship, allowing the transmission and establishment of the disease in the field [42]. Another factor that may have influenced the absence of transmission of fungi present in the seeds may be due to disinfection made with sodium hypochlorite 1%. It is known that the inhibitory effect of sodium hypochlorite has the ability to penetrate the microbial cell, destroying it [43]. The microorganisms were probably found in the external parts of the seeds, which allowed a decrease in the incidence at the time of the disinfestation. According to Botelho et al., (2008) [44], the fungi in the asepsis process have less survival capacity, however, for its total elimination, it is indispensable to consider several factors such as -location of the fungus in the seed, conditions in which the concentration of the product and immersion time in the solution. In general, this report is interesting and has contributed valuable information in research on sanity, pathogenicit, and transmission of Chia seeds, allowing to broaden the information contained in the literature and thus to subsidize future studies such as described in the present study.
IV. CONCLUSION Chia seeds from the municipality of Gurupi-TO showed a low incidence of fungi. There was no pathogenicity and transmissibility of the fungi detected in Chia seeds in seedlings.

International Journal of Advanced Engineering Research and Science (IJAERS)
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