Microstructure characterization of different types ofchlamydospores inDuddingtonia flagrans
Bobo Wang, Fenghui Wang, Youlei Li *, Kuizheng Cai *
(Medical College of Yan’ an University, Yan’an 716000, China)
*These authors contributed equally to this study.
*Correspondence: Kuizheng Cai, Medical College of Yan’an University, Yan’an 716000, China.
E-mail: ckz000@126.com
Phone: 86-0911-2650158
Fax: +86-0911-2650158
*Correspondence: Li You Lei, Medical College of Yan’an University, Yan’an 716000, China.
E-mail: liyoulei@yau.edu.cn
Phone: 86-0911-2650158
Fax: +86-0911-2650158
Abstract: The morphological and structural differences of different types of chlamydospore of Duddingtonia flagrans , a nematophagous fungus, were studied under light microscope and electron microscope to provide reference for the biological control of parasitic nematodiasis. In this study, D. flagrans isolate F088 dormant chlamydospore and non-dormant chlamydospore were selected as the research objects. The structural differences of these spores were observed by optical microscopy through lactol cotton blue, Trypan blue and MTT staining. FUN-1, DAPI and CFW staining were used to observe the metabolic activity, cell wall and nucleus differences of the two types of spores under fluorescence microscope. Ultrastructure of the two kinds of spores was observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Since lacto phenol cotton blue, trypan blue staining cannot distinguish dormant spores from dead spores, MTT assay was performed. Fluorescence microscopy observation showed that the cytoplasmic metabolic activity of non-dormant spores was stronger than that of dormant spores. The nucleus of dormant spores was bright blue, and their fluorescence was stronger than that of non-dormant spores. The cell wall of non-dormant spores produced stronger yellow-green fluorescence than that of dormant spores. Ultrastructural observation showed that there were globular protuberances on the surface of the two types of spores, but with no significant difference between them. The inner wall of dormant spore possesses a thick zona pellucida with high electron density which was significantly thicker than that of non-dormant spores, and their cytoplasm is also changed. In this study, the microstructure characteristics of dormant and non-dormant chlamydospores of D. flagrans fungi were preliminarily clarified, suggesting that the state of cell wall and intracellular materials were changed after spores entered to dormancy.
Keywords: Duddingtonia flagrans ; nematophagous fungi; dormant chlamydospore; non-dormant chlamydospore; metabolic activity; ultrastructure
Introduction
Nematophagous fungus Duddingtonia flagrans is a promising candidate for biological control of animal helminthic diseases. The chlamydospore of this fungus was produced very early in the growth process. Compared with other nematopgagous fungi that did not produce chlamydospore, this fungus could produce chlamydospore in three days and produced few conidia [1,2]. This fungus is the only species of chlamydospore producing among the nematopgagous fungi of the Orbiliaceae family [3]. In addition, the advantage of this fungus in the biological control of animal parasitic nematodes is that the double-walled chlamydospore added to the feed can resist the adverse environment of the digestive tract [4-7]. The spores then develop along with the worm eggs in the feces of the animal, the spores of the fungus begin to germinate, and the larvae that hatch further stimulate the mycelium of the fungus to specialize into feeding organs to feed on the larvae as nourishment for the fungus itself [6]. Due to the strong resistance of chlamydospore to harsh external environment, such as freezing and drying, chlamydospore as an effective component of feed additives, such as various formulation, lick blocks and powders, has good commercial value [8-10]. Both powder and pellet formulation can effectively reduce infectious larvae in pasture, and have obvious control effect on gastrointestinal nematodes in grazing horses, cattle, sheep, goats and other livestock [11-13].
The dormancy of spores is one of the most mysterious life forms because of their resistance to adverse conditions and their ability to remain in a state of hypometabolism (a sort of half-dead) for long periods of time, which might be the best form for the source of all life on Earth [14]. The study of fungal spore dormancy is much later than that of bacterial spore. There are still many questions to explain the phenomenon of fungal spore dormancy. D. flagrans is a species of fungi belonging to the Deuteromycotina. At present, no sexual stage has been found, and the chlamydospore is asexual.
These spores are often found dormant in nature or in biocontrol products [15]. And this dormancy is a deep endogenous dormancy, which is different from the exogenous dormancy of some fungi such asAspergillus conidium [16]. Endogenous dormancy spore cannot germinate even in the presence of sufficient nutrients such as water, carbon and nitrogen sources. At present, researchers pay more attention to the dormancy of some species of Aspergillus andTrichophyton rubrum conidium [17-20], and these fungi are mainly opportunistic pathogens and cause skin diseases in humans. However, there are relatively few literatures on the observation of the ultrastructure of fungal spores. Anjo et al. observed the structural characteristics of conidia of A. fumigatus at different culture stages by transmission electron microscopy [21]. Some studies observed the ultrastructure of the resting and germinating conidia ofA. ochraceus and the resting conidia of Botrytis cinerea [22,23]. The structure of dormant chlamydospore of fungi was less observed. Among these studies, Citiculo et al. observed the metabolic activity of chlamydospore at different days of age by fluorescence microscopy against the conditioned human pathogens Candida albicans and Candida Dublin [24]. Yan et al. observed the ultrastructure of dormant and non-dormant chlamydospore of M. oryzae [25]. In some reports, the interaction of D. flagranswith L3 of C. contortus was observed by SEM [26,27]. However, the morphological data of dormant and non-dormant of D. flagranschlamydospore are still lacking. In the practical application of biotechnology, producers encounter the problems of chlamydospore seed material inspection and final product quality inspection. Due to the existence of structural dormancy of chlamydospore in the product, it could not germinate on the basis of normal culture, so the conventional methods of spore incubation and colony counting could not reflect the actual number of live spores. Therefore, to fix this problem, it is necessary to find an effective method to distinguish dormant spores from dead spores by morphology. Meanwhile, it is also of great scientific significance to study the morphological and structural differences between dormant spores and non-dormant spores.
In this study, we aim to elucidate the morphological differences of different types of chlamydospore under light microscopy and electron microscopy. Firstly, the structural differences of dormant, non-dormant and dead spores were observed by optical microscopy with different staining methods. Secondly, the differences of metabolic activity, cell wall and nucleus between dormant and non-dormant spores were observed by fluorescence microscopy. Finally, the ultrastructure of the two types of spores was compared by scanning electron microscope and transmission electron microscope. The data obtained in this study provide a scientific basis for the application of D. flagrans in the biological control of animal parasitic nematodes and for understanding the dormancy mechanism of the fungal chlamydospore.