Macrophoma rot is a general term for fruit rots of Vitis spp. caused by the fungus Neofusicoccum ribis (syn. Botryosphaeria ribis) or closely related or renamed taxa, including Botryosphaeria dothidea. While mainly observed as a fruit pathogen of muscadine grape, the disease has recently been observed on bunch grapes in Virginia. Isolates (N = 835) were collected from Petit Manseng fruit clusters from seven Virginia vineyards in 2018 and 2019. A subset of these isolates was sequenced using three primer sets (ITS, RPB2, and EF). The preliminary result showed a single taxonomic strain of N. ribis. A controlled inoculation study of Petit Manseng clusters verified that infection could occur anytime between bloom and 2 weeks post-veraison; however, both the mean cluster incidence and the severity of Macrophoma rot did not differ from each other at any growth stage during the season. A season-long cluster exposure experiment showed that any amount of sun exposure significantly increased Macrophoma rot severity compared to shaded clusters, and that full sun exposure was associated with greatest rot severity. This finding contravenes current management recommendations for Macrophoma rot, and it raises yet unanswered questions as to why exposed clusters are more susceptible to Macrophoma rot than are shaded clusters. An in vitro fungicide assay study using nine fungicides identified captan, thiophanate-methyl, and tetraconazole as potential candidates for management of Macrophoma rot which need to be investigated further. / Master of Science in Life Sciences / Macrophoma rot is a general term for fruit rots of grapes caused by the pathogenic fungi in the family Botryosphaeriaceae. The rot is mainly observed on Muscadine grapes, but recently more cases were found on a wine grape cultivar Petit Manseng in Virginia. Macrophoma rot symptoms begin as dark brown, circular lesions on the surface of the berry and look similar to sunburn and other fruit rots. As the disease progresses, the lesion envelopes the entire berry and black fruiting bodies develop. Severe cases may lead to crop loss. The same group of pathogens is also associated with rots on other crops including apple, pear, olive, and kiwis. Very little is known about the disease cycle and the control of Macrophoma rot, therefore, an investigation into this fungal pathogen was needed. Multiple studies with the wine grape variety Petit Manseng were conducted during the 2018-2019 growing seasons, including a survey, leaf removal trial, and an inoculation study. Results showed that a species called Neofusicoccum ribis was found in vineyards across northern and central Virginia based on the genetic identification of fungal isolates collected at seven vineyards in those areas. Macrophoma symptoms were observed to be more prevalent and severe in more exposed clusters based on a leaf removal experiment. An artificial inoculation experiment revealed that grape clusters are susceptible to Neofusicoccum ribis at any time during the season. Based on the screening of nine fungicides, three chemicals (captan, thiophanate-methyl, and tetraconazole) showed promising results as possible management tools for Macrophoma rot. The knowledge collected will lead to an increase in understanding of this fungal pathogen and to further studies to manage Macrophoma rot.

Elevados valores de incidência da podridão apical da goiaba vêm sendo observados em levantamentos recentes no Estado de São Paulo. Entretanto, pouco se conhece sobre a etiologia e influência das variáveis ambientais no monociclo dessa doença. O objetivo desse trabalho foi identificar as espécies de Botryosphaeriaceae que causam a podridão apical e estudar as condições ambientais favoráveis, in vivo e in vitro, para o desenvolvimento do monociclo desses patógenos. Para a identificação das espécies, 56 isolados monospóricos foram obtidos das principais regiões produtoras de goiabas do Estado de São Paulo. Com auxílio da caracterização morfológica e de análises filogenéticas realizadas com dados de sequências de DNA da região ITS e β-tubulina foram identificadas as espécies Fusicoccum aesculi, Neofusicoccum parvum e Neofusicoccum ribis. O crescimento micelial dessas espécies foi avaliado sob as temperaturas de 10, 15, 20, 25, 30, 35 e 40 ºC. A germinação de conídios foi avaliada sob temperaturas de 10, 15, 20, 25, 30, 35 e 40 ºC com períodos de molhamentos de 4, 6, 12, 24 e 48 horas. Nos experimentos in vivo goiabas cv. Kumagai foram inoculadas com suspensões de conídios das três espécies identificadas com auxílio de ferimentos artificiais. Esses frutos foram mantidos sob as temperaturas de 15, 20, 25, 30 e 35 ºC com períodos de molhamento após a inoculação de 6, 12, 24 e 48 horas. Avaliou-se o período de incubação, o diâmetro da lesão ao sétimo dia após a inoculação e taxa de crescimento da lesão. A temperatura ótima para o crescimento micelial de N. ribis foi de 28,2 ºC e não houve diferença entre as temperaturas ótimas de N. parvum e F. aesculi, que foram de aproximadamente 31 ºC. A temperatura ótima para germinação dos conídios foi de 30 ºC para as três espécies. Houve incremento na germinação com o aumento do período de molhamento e na faixa de temperatura ótima encontram-se valores superiores a 70% de germinação a partir do molhamento de 6 horas. A espécie F. aesculi alcança elevadores valores de germinação a 40 ºC em períodos de molhamento superiores a 12 horas. A temperatura ótima para o crescimento da lesão foi de aproximadamente 30 ºC para as três espécies e o período molhamento de 48 horas proporcionou um aumento na severidade da doença quando comparado com o molhamento de 6 horas. Os menores períodos de incubação foram obtidos na temperatura de 30 ºC e com períodos de molhamento de 48 horas após a inoculação. Não houve diferença entre os valores das taxas de crescimento da lesão das três espécies e média das taxas das três espécies foi de 0,4. A doença se desenvolveu melhor em condições de temperaturas elevadas e períodos prolongados de umidade. Esse é o primeiro relato de Neofusicoccum parvum e Neofusicoccum ribis associados à podridão apical no Brasil. / Increased incidence of stylar-end rot in guava fruits have been observed in the São Paulo State - Brazil. However, little is known about its etiology and the influence of environment variables on the monocycle of this disease. This study aimed to identify Botryosphaeriaceae species that cause stylar-end rot in guava and to analyze the favorable environment conditions, in vitro and in vivo, for the monocycle development of this pathogen. We used 56 monosporic isolates from diseased fruit from guava producing regions from the São Paulo State - Brazil. The morphological and phylogenetic analyses performed with sequence data of the ITS region and β- tubulin allowed to identify the species Fusicoccum aesculi, Neofusicoccum parvum and Neofusicoccum ribis, as causal agents of the disease. The mycelial growth was evaluated at 10, 15, 20, 25, 30, 35 and 40 °C. Conidial germination was evaluated at 10, 15, 20, 25, 30, 35 and 40 °C with wetness periods of 4, 6, 12, 24 and 48 hours. In the in vivo experiments, guavas cv. Kumagai were inoculated with conidial suspensions of the three species identified by artificial wounds. The fruits were kept at 15, 20, 25, 30 and 35 °C with wetness periods of 6, 12, 24 and 48 hours after inoculation. We evaluated the incubation period, the lesion diameter on the seventh day after inoculation and the lesion growth rate. The optimum temperature for mycelial growth of N. ribis was 28.2 °C and there was no difference between the optimal temperatures (approximately 31 ºC) for N. parvum and F. aesculi. The optimal temperature for conidial germination was 30 ºC for the three species. There was an increase in germination with the increase of the wetness period. Within the optimal temperature range, values were higher 70% of germination after a wetness period of 6 hours. F. aesculi reached high germination values at 40 °C in wetness periods exceeding 12 hours. The optimal temperature for lesion growth rate was approximately 30 °C for the three pathogens and the wetness period of 48 hours caused higher disease severity as compared with the wetness period of 6 hours. The shortest incubation periods were obtained at 30 ºC with wetness periods of 48 hours. There was no difference between lesion growth rate for the three species and the average rate for three pathogens was 0.4/day. The best conditions for the disease development are high temperatures and prolonged periods of moisture. This is the first report of Neofusicoccum parvum and Neofusicoccum ribis associated with stylarend rot of guava fruits in Brazil.

Elevados valores de incidência da podridão apical da goiaba vêm sendo observados em levantamentos recentes no Estado de São Paulo. Entretanto, pouco se conhece sobre a etiologia e influência das variáveis ambientais no monociclo dessa doença. O objetivo desse trabalho foi identificar as espécies de Botryosphaeriaceae que causam a podridão apical e estudar as condições ambientais favoráveis, in vivo e in vitro, para o desenvolvimento do monociclo desses patógenos. Para a identificação das espécies, 56 isolados monospóricos foram obtidos das principais regiões produtoras de goiabas do Estado de São Paulo. Com auxílio da caracterização morfológica e de análises filogenéticas realizadas com dados de sequências de DNA da região ITS e β-tubulina foram identificadas as espécies Fusicoccum aesculi, Neofusicoccum parvum e Neofusicoccum ribis. O crescimento micelial dessas espécies foi avaliado sob as temperaturas de 10, 15, 20, 25, 30, 35 e 40 ºC. A germinação de conídios foi avaliada sob temperaturas de 10, 15, 20, 25, 30, 35 e 40 ºC com períodos de molhamentos de 4, 6, 12, 24 e 48 horas. Nos experimentos in vivo goiabas cv. Kumagai foram inoculadas com suspensões de conídios das três espécies identificadas com auxílio de ferimentos artificiais. Esses frutos foram mantidos sob as temperaturas de 15, 20, 25, 30 e 35 ºC com períodos de molhamento após a inoculação de 6, 12, 24 e 48 horas. Avaliou-se o período de incubação, o diâmetro da lesão ao sétimo dia após a inoculação e taxa de crescimento da lesão. A temperatura ótima para o crescimento micelial de N. ribis foi de 28,2 ºC e não houve diferença entre as temperaturas ótimas de N. parvum e F. aesculi, que foram de aproximadamente 31 ºC. A temperatura ótima para germinação dos conídios foi de 30 ºC para as três espécies. Houve incremento na germinação com o aumento do período de molhamento e na faixa de temperatura ótima encontram-se valores superiores a 70% de germinação a partir do molhamento de 6 horas. A espécie F. aesculi alcança elevadores valores de germinação a 40 ºC em períodos de molhamento superiores a 12 horas. A temperatura ótima para o crescimento da lesão foi de aproximadamente 30 ºC para as três espécies e o período molhamento de 48 horas proporcionou um aumento na severidade da doença quando comparado com o molhamento de 6 horas. Os menores períodos de incubação foram obtidos na temperatura de 30 ºC e com períodos de molhamento de 48 horas após a inoculação. Não houve diferença entre os valores das taxas de crescimento da lesão das três espécies e média das taxas das três espécies foi de 0,4. A doença se desenvolveu melhor em condições de temperaturas elevadas e períodos prolongados de umidade. Esse é o primeiro relato de Neofusicoccum parvum e Neofusicoccum ribis associados à podridão apical no Brasil. / Increased incidence of stylar-end rot in guava fruits have been observed in the São Paulo State - Brazil. However, little is known about its etiology and the influence of environment variables on the monocycle of this disease. This study aimed to identify Botryosphaeriaceae species that cause stylar-end rot in guava and to analyze the favorable environment conditions, in vitro and in vivo, for the monocycle development of this pathogen. We used 56 monosporic isolates from diseased fruit from guava producing regions from the São Paulo State - Brazil. The morphological and phylogenetic analyses performed with sequence data of the ITS region and β- tubulin allowed to identify the species Fusicoccum aesculi, Neofusicoccum parvum and Neofusicoccum ribis, as causal agents of the disease. The mycelial growth was evaluated at 10, 15, 20, 25, 30, 35 and 40 °C. Conidial germination was evaluated at 10, 15, 20, 25, 30, 35 and 40 °C with wetness periods of 4, 6, 12, 24 and 48 hours. In the in vivo experiments, guavas cv. Kumagai were inoculated with conidial suspensions of the three species identified by artificial wounds. The fruits were kept at 15, 20, 25, 30 and 35 °C with wetness periods of 6, 12, 24 and 48 hours after inoculation. We evaluated the incubation period, the lesion diameter on the seventh day after inoculation and the lesion growth rate. The optimum temperature for mycelial growth of N. ribis was 28.2 °C and there was no difference between the optimal temperatures (approximately 31 ºC) for N. parvum and F. aesculi. The optimal temperature for conidial germination was 30 ºC for the three species. There was an increase in germination with the increase of the wetness period. Within the optimal temperature range, values were higher 70% of germination after a wetness period of 6 hours. F. aesculi reached high germination values at 40 °C in wetness periods exceeding 12 hours. The optimal temperature for lesion growth rate was approximately 30 °C for the three pathogens and the wetness period of 48 hours caused higher disease severity as compared with the wetness period of 6 hours. The shortest incubation periods were obtained at 30 ºC with wetness periods of 48 hours. There was no difference between lesion growth rate for the three species and the average rate for three pathogens was 0.4/day. The best conditions for the disease development are high temperatures and prolonged periods of moisture. This is the first report of Neofusicoccum parvum and Neofusicoccum ribis associated with stylarend rot of guava fruits in Brazil.