The First report of Nigrospora sphaerica Associated with Heliocarpus americanus Seeds in Brazil

Abstract

Heliocarpus americanus is a fast-growing native tree, excellent for recovering degraded areas and its wood can be used as firewood or in the production of charcoal. For its use it is necessary identify the fungus will cause diseases. This work aimed to make the first report of the occurrence of the fungus Nigrospora sphaerica in seeds of H. americanus in Brazil. The fungus was detected in a batch of seeds collected from the native species. The fungus were analyzed by the Blotter test. Then, the fungus was isolated from the seeds and it characterized by morphology of colony and conidia and, molecular tests, what confirmed the identity of the pathogen. To conclude the study, the Koch’s postulates test was performed, where it was observed its transmission of seeds to H. americanus seedlings, elucidating the fungal damage in the seeds and later seedlings of this species. Keyworks: seeds forest, seeds phatolology, forest physiology 1Universidade Tecnológica Federal do Paraná (UTFP), Agronomia, Dois Vizinhos, PR, Brasil 2Universidade Tecnológica Federal do Paraná (UTFP), Agronomia, Toledo, PR, Brasil Heliocarpus americanus L. is a dioecious native plant that is considered an excellent species for recovering degraded areas because of its rapid growth and economic value in producing wood that can be used as firewood and for charcoal production (Lorenzi, 1992). Thus, this species is a good option for cultivation. To produce healthy and good quality seedlings of this species, seeds with good physiological and sanitary properties should be used. Phytopathogenic fungi are the primary cause of diseases in plant species in forests and nurseries, and many of these fungi remain unidentified, making them difficult to control. To our knowledge, this is the first study reporting the occurrence of the fungus Nigrospora sphaerica in seeds of H. americanus in Brazil. In March 2017, seeds were collected directly from the native trees in Engenheiro Beltrão, Paraná, Brazil (23°47’49”S; 52°16’08”W). Sanitary quality surveys of the seeds (Silva et al., 2016) revealed that 40% of the samples had a fungal infection (Figure 1A). This incidence is significant given the risk of transporting phytopathogens associated with the seeds, which represents a mechanism for the spread of pathogens both locally and to new areas. The infected seeds were surface disinfected in 0.5% NaClO solution for 1 minute and then washed three times in sterile water. Subsequently, the seeds were placed on potato dextrose agar (PDA) plates and incubated at 24 ± 1°C with a photoperiod of 12 hours. The fungi were recovered on the 7th day of incubation. The fungal colonies exhibited filamentous black coloration, a flat raised surface, and filiform borders (Figure 1B). After 10 days of mycelial growth, black, spherical, and unicellular conidia with an average diameter of 18.7 μm (range, 15.5–22 μm) were identified under an optical microscope (Figure 1C). The conidia were present on the hyaline vesicles located at the tip of the short and sparsely branched conidiophores similar to those described by Han et al. (2019). Based on these morphological characteristics, we identified the colonies as N. sphaerica (Sacc.) E.W. Mason. For molecular identification, we extracted fungal DNA directly