Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil

Abstract

Black pod disease, caused by <i>Phytophthora</i> species, is among the main limiting factors of cacao (<i>Theobroma cacao</i> L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by <i>Phytophthora capsici</i>, <i>Phytophthora citrophthora</i>, <i>Phytophthora heveae</i>, and <i>Phytophthora palmivora</i>. To assess the diversity of <i>Phytophthora</i> species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as <i>P. citrophthora</i> and <i>P. palmivora</i> were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome <i>c</i> oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting <i>Phytophthora</i> isolates. The morphological characterization and evaluation of the mycelial growth rates for the <i>Phytophthora</i> isolates were performed <i>in vitro</i>. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as <i>P. palmivora</i> (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in <i>Phytophthora</i> clade 2. Therefore, these isolates are considered as a new species herein referred to as <i>Phytophthora theobromicola</i> sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The <i>P. palmivora</i> isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of <i>P. capsici</i>, but no oogonia/antheridia were observed when <i>P. theobromicola</i> was paired with the different tester strains. The <i>P. theobromicola</i> and <i>P. citrophthora</i> isolates showed higher mycelial growth rates, when compared to <i>P. palmivora</i>, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both <i>Phytophthora</i> species, with <i>P. theobromicola</i> inducing higher rates of necrotic lesion expansion, when compared to <i>P. palmivora</i>. Here, two <i>Phytophthora</i> species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed <i>P. theobromicola</i> seems to be prevalent in field conditions. This is the first report of <i>P. theobromicola</i> on <i>T. cacao</i>. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to <i>Phytophthora</i>.