Etiology, transmission and pathogenicity of fungi associated with Cagaita (Eugenia dysenterica) and Tingui (Magonia pubescens)

Cagaita (Eugenia dysenterica) and Tingui (Magona pubescens) are two species of trees native to the Cerrado, with sociological, economic, and medicinal importance, besides to being used in the recovery of degraded areas. The incidence of plant diseases, as well as the mycoflora associated with their seeds, may represent a possible threat to the survival of the species. The cultivation of trees in the Cerrado Biome, as well as the diseases associated with them, are still poorly studied. The present work aimed to evaluate the mycoflora associated with Cagaita and Tingui seeds, the transmission and pathogenicity of these fungi to plants. The seeds were collected from mother trees located in the municipality of Gurupi and Figueirópolis in the state of Tocantins, Brazil. The blotter-test method was used to evaluate the mycoflora associated with the seeds of both species, with and without asepsis. The fungal incidence per seed was evaluated with the aid of a stereoscopic and optical microscope. The potentially pathogenic fungi identified in the sanitary analysis of the seeds and in the transmission, test were inoculated in the plants in order to confirm Koch's postulates. The transmission test was carried out from non-disinfested seeds until the appearance of symptoms in the seedlings. A total of 11 fungi associated with the seeds of both species were identified. The genus Curvularia showed pathogenicity, causing spots on Cagaita leaves, being also transmitted from seeds to seedlings. The Fusarium genus showed pathogenicity in Tingui seedlings, causing damping-off and wilting. Colletotrichum sp. caused leaf spots on both species and fruit spots on Cagaita.


Introduction
The Cerrado is a savanna formation that occupies most of Brazil's Central Plateau. With a surface of approximately 2 million km², equivalent to 22% of the Brazilian territory (Ministério do Meio Ambiente, 2019). Although it is still a little studied biome, compared to the Amazon, it is internationally considered a biodiversity hotspot (Critical Ecosystem Partnership Fundation, 2017). Most plant species have high economic and ecological potential. In addition to its social importance, it often complements the diet and serves as a source of medicines, fiber, wood for construction and energy for the local inhabitants.
The cerrado is considered the richest savanna in the world in terms of biodiversity, besides having a high level of endemism (Klink & Machado, 2005).
Eugenia dysenterica DC., known as Cagaita or Cagaiteira is a tree in the Myrtaceae family. It is a characteristic tree of the cerrado, being found in Bahia, Minas Gerais, Goiás, Tocantins, São Paulo and Mato Grosso do Sul. Its distribution is irregular, being abundant in certain places and absent in others (Lorenzi, 1998). The flowering of Cagaita occurs in September, together with the recovery of the foliage. Fruiting coincides with the first rains, that is, from October to November (Camilo et al., 2013). Cagaita is indicated for the recovery of degraded areas due to the following characteristics: heliophilous, xerophytic (Lorenzi, 1998) and zoochorous (Sano et al., 1995), melliferous (Simeão et al., 2015), low nutritional requirements (Duboc & Guerrini, 2007). In addition, Cagaita represents a high economic potential for the Cerrado regions, however it is poorly developed due to lack of consumer acceptance and difficulty in seedling production (Ministério do Meio Ambiente, 2016).
Magonia pubescens St. Hill, commonly called Tingui, Timbó, Tingui-do-Cerrado, Cuitê, Timbopeba, or Tangui, belongs to the family of Sapindaceae. Tingui is found in the cerrado, both in primary and secondary formations, from Ceará to Minas Gerais, passing through Goiás, Tocantins, Mato Grosso and Mato Grosso do Sul. Flowering occurs from August to September, and fruit maturation can take a year, so seed production will occur in August, September of the following year. It is a heliophilous, pioneer, deciduous, selective xerophytic tree species (LORENZI, 2008). However, the seedlings support several levels of shading, from full sun to 70% shading . This species is recommended in the recovery of degraded areas, in particular due to its colonization by arbuscular mycorrhizal fungi (Scabora et al., 2011).
It is known that seed germination, survival and mortality rate are the first indicators evaluated in a process of recovery of degraded areas (Palma & Laurance, 2015). The basis of successful planting of native species depends on several factors, including the sanitary quality of the seeds (Lima et al., 2014). Due to the seasonality of fruiting (Kuhlmann, 2012) and the difficulty of monitoring and harvesting , seeds from native trees are still poorly studied. Furthermore, the incidence of plant diseases, as well as the mycoflora associated with their seeds, may represent a possible threat to the survival of native species, which must be multiplied before being used in degraded areas.
Given the above, this study aimed to evaluate the mycoflora associated with Cagaita and Tingui seeds, the transmission and pathogenicity of these fungi to these plants intended for the recovery of degraded areas.

Methodology
The experiments were conducted at the Phytopathology Laboratory of the Federal University of Tocantins, Gurupi-TO campus.

Origin of seeds and place of experiment
The seeds of both species were collected (Table 1) between September and October 2018, at the UFT de Gurupi campus and its surroundings. According to the species, a collection method was chosen. The seeds were removed from the fruits, dried in the shade in a dry and ventilated place, and later placed in a paper bag and stored in a refrigerator at 5 o C.  Source: Authors.

Transport of fungi associated with seeds
The method used to survey fungal populations associated with the seeds was the filter paper method or "Blotter Test" (BRASIL, 2009). An acrylic Gerbox box, disinfected with 50% alcohol and 1% Sodium Hypochlorite, was used, containing two sheets of filter paper sterilized by autoclave and moistened with sterile distilled water.
The seeds were subjected to treatments, with and without asepsis, performed in the following sequence: seeds immersed in 50% alcohol solution for 40 seconds, followed by 1% sodium hypochlorite for 40 seconds. After disinfestation, they were washed twice in sterile distilled water and left to dry at room temperature.
200 seeds per species were used and divided into 5 repetitions of 40 seeds. Due to the unequal size of the seeds, each repetition was composed of four gerboxes for Cagaita and eight for Tingui. Subsequently, the boxes were placed in a growth chamber, type BOD, at a temperature of 25 °C and a photoperiod of 12 hours for a period of five to seven days. After this period, each seed was examined with the aid of a magnifying glass and optical microscope, where slides were prepared for Research, Society and Development, v. 11, n. 3, e55211326975, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26975 visualization of fungal structures (conidia and conidiophores). The initial identification of the genres was made with the help of specialized literature (Watanabe, 2010). The identified fungi were isolated and cultivated in potato-dextrose-agar (PDA) culture medium with antibiotic (Amoxilin) to limit the growth of bacteria.
The experimental design used was completely randomized, with five replications. The treatments were arranged in a 2x2 factorial scheme (seed and species treatment). For variance analysis, the incidence values of each fungal genus were transformed by the following formula: f(x) = arcsine (square root (x)), which: x = incidence of the genera in the seeds and f(x) = Transformed variable.
The transformed variable of each fungal genus was submitted to the ANOVA test for the model that considered the effect of the treatments. The means were compared by the Scott-Knott Test at 5% significance. For data analysis, the R Statistical Analysis System version 3.5.2 was used.

Transmissibility of fungi from seeds to seedlings
The seeds were sown without asepsis and without fungicide treatment, in order to evaluate the natural incidence of the fungi and their transmission to the plants. After sowing, it was sought to maintain soil moisture, so that the environment was favorable for transmission. During the growth of the seedlings, the appearance of the first symptoms of diseases were observed. To confirm Koch's Postulates, plant fragments that showed symptoms were detached for isolation. The plant material was collected and disinfected in the following sequence: a 50% alcohol solution for 40 seconds, followed by 1% sodium hypochlorite for 40 seconds and finally washed in sterile water. Then, the tissues were incubated in petri dishes with PDA medium. Subsequently, the petri dishes were placed in a growth chamber, type BOD, at a temperature of 25 °C and a photoperiod of 12 hours for a period of five to seven days. The plant material was observed with a magnifying glass and an optical microscope to verify the presence of fungal structures and aided by specialized literature.

Pathogenicity of fungi on seedlings and fruits
According to the physiology of each species, different treatments were made to overcome dormancy. For Cagaita, there was seed dormancy of tegumentary and biochemical origin. According to some authors, the integument of Cagaita seeds does not prevent water absorption, but limits gas exchange (Martinotto et al., 2008;Silveira et al., 2013). With scarification or complete suppression of the integument, the percentage of germination reaches 88%, therefore, the integument is removed with the help of a sterile stylet. Tingui seeds have a high germination rate without treatment, therefore, they were sown without any treatment. The seeds were sown in a black plastic bag (9 x 15 cm) containing a mixture of sterilized sand mixed with organic compost. The seedlings were irrigated daily. For each species, the fungi isolated during the sanitary analysis were inoculated in the aerial parts or/and in the soil according to the mode of infection of each fungal species. When the seedlings presented more than two definitive leaves, sprays were made with conidia solutions at a concentration of 2x106 spores mL -1 . Suspensions were prepared from colonies of fungi previously isolated in PDA medium. The inoculated seedlings were placed in a moist and dark chamber for 72 h. After this period, the seedlings were removed from the moist chamber and observed until the appearance of the first symptoms. To confirm pathogenicity, Koch's Postulates were performed. Table 2 shows the average incidence of fungal genera found in Cagaita and Tingui seeds. According to the results obtained, a total of nine genders were identified. Research, Society andDevelopment, v. 11, n. 3, e55211326975, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26975 There was a significant difference between the species of fungi in general (Pr = 0.0010 < 0.05), for Cagaita (Pr = 0.0000 < 0.05) and for Tingui (Pr = 0.0000 < 0.05). 05). Table 2 -Mean incidence (%) of fungal genera associated with Cagaita and Tingui seeds submitted to treatments with asepsis (CA) and without asepsis (SA) 1 . 18.4 a 12.7 a 12,9 1 Means followed by the same letter has no significant differences according to the Scott-Knott Test (P < 0.05), with capital letters relating to each column and lowercase letters relating to each row. 2 Average fungal incidence per species. 3 Average incidence per fungal gender. Source: Authors.

Gender
Of the fungal genera identified, four of them were common associated with the two species of trees: Cladosporium, Aspergillus, Penicillium and Fusarium. These same fungi were also found in Cerrado plant species in studies developed by Pinheiro et al. (2016). According to studies developed by Watanabe (2010), these fungi are very frequent because they are associated with plants. It is known that some fungi found associated with seeds can be considered as being external saprophytes).
There were no significant differences between Cagaita and Tingui in terms of total fungal incidence (Pr = 0.5212 > 0.05). But there were differences between the associated fungal genders. Eight fungi associated with Cagaita seeds were found, Nine genera associated with Tingui seeds were found. In addition to the common genera already mentioned, Periconia and Trichoderma were also identified.
The asepsis had a significant effect on the elimination of some fungi, decreasing the incidence in seeds (Pr = 0.0146 < 0.05). Whereby, in the seeds of the forest species studied, the treatment without asepsis had an average incidence of 24.96% and, in the treatment with asepsis, there was an incidence of 6.20%. Thus, it can be affirmed that the asepsis of the seeds significantly reduced the fungal incidence in the seeds of Cagaita and Tingui (Table 2).
For Cagaita seeds, the fungal incidence in seeds with asepsis was 5.12%, and 31.75% without asepsis. It can be observed the same fungi associated with the seeds in both treatments, that is, the asepsis did not eliminate the fungi, however, there was a decrease in their incidence. The asepsis can eliminate part of the inoculum (spores, hyphae fragments) and thus Research, Society and Development, v. 11, n. 3, e55211326975, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i3.26975 6 limit the development of fungi. According to Pinheiro (2016), this method acts on the surface of the seeds, protecting them from some pathogens. However, we have to consider that the treatment with alcohol and sodium hypochlorite does not present systemic action on the seeds, so that some more resistant fungi or those located in the more internal layers of the seeds can survive to the treatment.
However, unlike Cagaita, the specific diversity has also changed. Three fungi, two of them Aspergillus sp. and Periconia sp., were no longer found after asepsis. Some studies had similar results with other forestry species, where asepsis eliminated fungal species (Muniz et al., 2007). However, two other genera, absent in the treatment without asepsis, were found in the seeds with asepsis, being them, Cladosporium and Trichoderma. The Tingui seeds have external protective layers that, in contact with water, produce a gel composed of cellulosic oligosaccharides (Gorin et al., 1996), divided into five regions of several composition (Oliveira et al., 2001). This gel has potential antifungal activity, in particular against Penicillium, Aspergillus and Hormodendrum sp. Studies demonstrate the potential use of powder covering Tingui seeds as products for pelleting commercial seeds, as their characteristics are similar to HPMC (hydroxypropylmethylcellulose), in addition to having an antifungal activity (Teixeira, 2007). Thus, the alcohol and sodium hypochlorite, used in asepsis, may have denatured, or altered the protective gel of the seeds, which could explain these differences in the fungal population.
The fungi with larger frequency in Cagaita and Tingui were Aspergillus niger and Penicillium sp., with an incidence of 31.11% and 46.8% on seeds, respectively. Furthermore, it was observed that the asepsis did not eliminate these fungi that were associated with the seeds of both species, even after the disinfestation. These two fungi are usually found in closed environments of seed storage, so they are called storage fungi. Consequently, they may come from external contamination. The so-called storage fungi may not be phytopathogenic to plants, however, they can cause damage to the seeds (Dhingra et al., 2003). Under not suitable conditions, such as humidity and high temperature, the "storage" fungi can quickly degrade seeds and impair the seedling germination and emergence, in addition to producing toxic substances.
In particular, the seeds with a high-water content and sensitive to desiccation suffer more from saprophytic fungi.
Despite being generalist molds, these fungi can impair the seed storage, especially in tropical climates. The Cagaita seeds are recalcitrant, that is, they cannot withstand a long period of storage, desiccation and very low temperatures (Delgado & Barbedo, 2012). They present a high-water content, between 47 and 53%. When the water content of the seeds dropped below 18-22%, the seeds lost their viability. The ideal for its storage is inside plastic bags in a cold chamber at 10 °C with 60% humidity (Andrade et al., 2003). These conditions favor the development of fungi associated with the seeds and their deterioration, as shown in species close to Cagaita, Eugenia brasiliensis, E. pyrifomis and E. uniflora (Oliveira et al., 2011).
Preferably, the Cagaita seeds should be collected in the soil than in the tree, for a better germination rate, however, this can increase the risk of contamination by fungi. Certain fungi may be associated with the seeds, and remain dormant until the fruit maturation (Singh, 2011).
The orthodox species tend to have a low water content, which limits the fungi development and attack. Unlike to the recalcitrant species that need to be kept with high water content, presenting greater colonization by so-called field fungi, such as Fusarium (Schmidt, 2007). The Tingui is an orthodox species (Consolaro et al., 2019). Its seeds have an average water content of 6% (Arantes et al., 2017). However, storage for up to 90 days is recommended, as it is after this period that the germination rate decreases and the seeds lose vigor (Jeromini et al., 2015).
The fungi found in the sanitary analysis of Cagaita and Tingui seeds can seriously compromise the storage of these species. Most Cerrado tree species produce seeds once a year and usually at the end of the dry season. These characteristics, together with the need to wait for the rainy season to the planting of species, require a storage period, even short, which can be compromised by the fungi identified in this work. The tropical climatic condition can, however, favor the degradation of seeds and, consequently, compromise their planting.

Transmission
In the transmission study, from seeds without disinfestation, arise in Cataiga seedlings, initially, yellow-brown leaf spots, which evolved into dark necrosis (Figure 1). It is worth noting, that in the evaluated period, during four months, these lesions were restricted to adult leaves that, over time, did not cause the death of the plants. This symptom type has already been reported by other authors, in nursery conditions (Martinotto et al., 2008).
From the leaf lesions, the isolations of necrotic tissues showed the presence of several associated fungi (Table 3).  Source: Authors.
The Tingui seedlings showed different symptoms, including stains on the edges of the leaves that progressed to the necrosis. In addition, some plants wilted with the dry leaves until death (Figure 2).
After isolating the lesions from different plant tissues, some associated fungi were found, as shown in Table 4.  Of the fungal genera found, Cladosporium and Fusarium had already been detected associated with Tingui seeds.
Only the Colletotrichum had not been observed in the seeds. In the same way that it appeared in the Cagaita seeds, this genus may have been transmitted by seeds, but with a very low incidence, or it was transmitted by the environment to the plant.
Although this genus was found associated with leaf spots, this fungus was not detected in the respective species seeds.

Pathogenicity
Of the eight fungal genera detected during the sanitary analysis of the seeds, or isolated from lesions on seedlings, in the transmission test, some of them were tested for pathogenicity in Cagaita plants, as can be seen in Table 5. The seedlings inoculated with Curvularia showed leaf spots after one month. The spots, irregularly shaped, started at the edges of the leaves and progressed to the center (Figure 3a). From these spots, the Curvularia were isolated again, which confirms the Koch's postulates. Even being pathogenic and associated with Cagaita seeds, the Curvularia was not observed during the transmissibility test. It is thus noted that for transmission, even for potentially pathogenic fungi, the inoculum transported via seed depends, fundamentally, on the cultivated species (resistance), environmental conditions (ambient and soil humidity, temperature, wind, rain and light), inoculum (viability, location in the seed), cultivation practices (soil type, pH, plant population, planting depth and planting season, fertilization), inoculum survival, soil and seed microflora, among others (Mayer et al., 2001).
The seedlings inoculated with Colletotrichum showed leaf spots, starting at the periphery, leading to total necrosis, after 4 days (Figure 3b). From these spots, Colletotrichum was isolated again, which allowed the confirmation of the Koch's postulates. In addition to the leaves, Colletotrichum was also inoculated in healthy Cagaita fruits. The pathogen caused rot symptoms on the fruits, 3 days after inoculation. Therefore, spores of Colletotrichum sp. were inoculated in healthy Cagaita fruits in order to test its pathogenicity.
After three days, rot symptoms with spots appeared on the fruits (Figure 3b). From these spots, the genus Colletrotrichum was identified under the microscope.
Colletotrichum gloesporioides is a pathogen of species close to Cagaita, such as Araça-boi (Eugenia stipitata), causing lesions on the fruits (Sterling-Cuellar et al., 2005). The Colletotrichum species are recognized as pathogens of a broad spectrum, that is, they can attack different plants, in particular their fruits (Phoulivong, 2012).
Of the fungi isolated, only the genera Colletotrichum and Fusarium will cause symptoms of diseases in seedlings of Tingui, confirming their pathogenicity, as can be seen in Table 6. The pathogenicity was confirmed through the Koch's Postulates. Colletotrichum caused leaf spots that appeared four days after inoculation (Figure 4a), while Fusarium sp. created the wilting and death of the seedlings (Figure 4b). According to the literature consulted, there were no records of these fungi as being pathogenic to the Magonia pusbescens plant.  Source: Authors.
The seedlings inoculated with Colletotrichum showed leaf spots after four days. The spots, of round shape and brown, started at the edges of the leaves and progressed to the center (Figure 4a). From these spots, the Colletotrichum sp. was isolated again, which confirmed Koch's postulates.
These results show the importance of studying tree species native from the Cerrado. In particular, the nursery diseases can quickly spread and compromise the final planting.

Conclusion
There was diversity of microflora associated with Cagaita and Tingui seeds. The fungi with the highest incidence in the seeds of these species are Penicillium sp. and Aspergillus niger, which were not eliminated by asepsis.
The asepsis can be used to reduce the incidence of fungi in Cagaita seeds. However, in Tingui seeds, even with a decrease in fungal incidence with this treatment, it is not recommended, because Tingui seeds have protective and antifungal layers, and the asepsis may compromise these protections.
The fungi identified as a pathogen for Cagaita were Curvularia sp., that caused leaf spot. The fungus Colletotrichum sp. also caused pathogenicity in Cagaita, both in leaves and fruits.
For Tingui, the fungi Colletotrichum sp., causing leaf spots, and Fusarium sp., causing the seedlings to wilt to death, were pathogenic.
More studies are also needed on the health, storage and alternative control of fungi associated with forest seeds and seedlings. Fungi present in seeds can cause lesions that result in the weakening or death of plant species used in the recovery of degraded areas such as Cagaita and Tingui.