Department of Genetics, Universidade Federal do Paraná, CEP 81531-980, Curitiba, PR, Brazil.
Department of Basic Pathology, Universidade Federal do Paraná, CEP 81531-980, Curitiba, PR, Brazil.
Microbiol Res. 2019 Sep;226:27-33. doi: 10.1016/j.micres.2019.05.005. Epub 2019 May 28.
Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.
采后果实脱落（PFD）主要由炭疽菌（Colletotrichum abscissum）引起，如果气候条件有利，可导致高达 80%的果实损失。根据文献记载，其他炭疽菌属物种利用不同的策略来定植宿主：细胞内半活体营养型或皮下壁内坏死营养型定植。然而，到目前为止，对于炭疽菌属，仅描述了坏死阶段，PFD 疾病周期中的某些方面仍不清楚。为了更好地了解疾病周期，可以应用显微镜研究。然而，即使使用表达绿色荧光蛋白（eGFP）的菌株，由于柑橘叶片的自发荧光，结果也不清楚。为了解决这个问题并研究炭疽菌属与柑橘的相互作用，我们使用了脱色和染色方法，我们观察到，即使在接种前进行损伤处理，炭疽菌属也不会定植相邻组织。显然，在叶片中，真菌仅利用人工损伤中暴露的营养物质进行生长，然后产生大量孢子。然而，在花朵中，炭疽菌属在接种后 12 小时就穿透并定植了花瓣组织。在感染的早期阶段，我们观察到初级活体营养菌丝的发育，表明该物种为半活体营养真菌，在花朵定植期间有一个短暂的活体营养阶段，随后是主要的坏死营养阶段。总之，使用炭疽菌属的 eGFP 菌株和一种不同的脱色和染色方法，使我们能够更好地理解这种柑橘病原菌用于定植宿主的形态和机制。
