Detection of Mycoplasmalike Organism of Paulownia Witches′-Broom with DAPI Fluorescence Microscopy

Paulownia witches’-broom infected by mycoplasmalike organism (MLO) has been developed several cytochemical methods for diagnosis. These methods all based on the special stain reactions or abnormal fluorescence in groups of infected sieve elements as a diseased symptom,. not really on the direct detection of MLO under light microscope. This paper deals with the demonstration of MLO specific white fluorescence after DAPI staining with GMA sections of diseased young stems. Such fluorescence was absent in sections from health plants. The results were confirmed by the ulrrastrueture of MLO and the structure of sieve elements showing from PAS-TBO stained GMA sections. The described method may not only be used in accurate diagnosis of MLO diseased in different plants, but is also worth in the studies of MLO distribution in plants, MLO dynamics in plant resting stage and MLO transmission to support the theoretical basis for protection.     

Genome-Wide Identification and Analysis of MicroRNAs Involved in Witches’-Broom Phytoplasma Response in Ziziphus jujuba

MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches’-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches’-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches’-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches’-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5’-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches’-broom disease in Z. jujuba.     

Specific In Situ Visualization of the Pathogenic Endophytic Fungus Aciculosporium take,the Cause of Witches’ Broom in Bamboo

The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches’ broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA of A. take was used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate that A. take grows endophytically, particularly in the shoot apical meristem of the host. The location of A. take hyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.The endophytic fungus Aciculosporium take (Ascomycota; Clavicipitaceae) causes continuous shoot growth, but the host bamboo plant maintains normal leaf arrangement and branching pattern (28, 29). This fungus produces the plant hormone auxin, indole-3-acetic acid (IAA) in culture (28). Hypersynthesis of auxins by pathogenic microorganisms has often been associated with diseases in which hyperplasia is exhibited by the host plant (31). However, the well-regulated morphological changes of the A. take-colonized bamboo cannot be explained by a mere hormonal imbalance. The symptom of A. take-colonized bamboo is characterized by the successive growth of thin phytomers (developmental modules consisting of a node, a stem segment, a leaf with a sheath, and a lateral bud) from a normal shoot apex (Fig. 1a and b). Diseased shoots continue to grow without branching and have extraordinarily small leaves. Sometimes, a diseased shoot develops 30 to 40 leaves, whereas healthy shoots develop only 3 to 5 leaves (25). When the conidiostroma is formed at the apex, the shoots cease to grow, and lateral buds initiate outgrowth (Fig. ​(Fig.1c).1c). This process occurs repeatedly and eventually results in a typical witches’ broom morphology after a few years (Fig. 1d and e). Excluding stroma formation, the fungus presents no external signs of its presence on the diseased shoots. Accurate localization of the growing hyphae would aid in establishing the process of symptom development.Open in a separate windowFIG. 1.Bamboo shoot colonized by the endophytic fungus Aciculosporium take. (a to c) Schematic of witches’ broom symptom development on a bamboo species of Phyllostachys. (a) The normal shoot ceased to grow after three to five leaves developed. (b) An A. take-colonized shoot grew out with extraordinarily small leaves but maintained normal leaf arrangement. The shoot continued to grow with successive thin phytomers. (c) After stromata (arrows) were formed at the shoot apex, the lateral buds began to grow but maintained a normal branching pattern. This sequence repeats. (d to f) Bamboo (P. pubescens) shoot colonized by A. take. (d) Early symptom. Diseased shoots (DS) have leaves that are much smaller than normal leaves (NL). Bar, 1 cm. (e) Developed symptom shows typical witches’ broom appearance. Bar, 1 cm. (f) Ascostroma (AS) is formed on a conidiostroma (CS) at the apex of the diseased shoot. The conidiostroma is surrounded by a sheath. Bar, 1 mm.Many fungal endophyte species have been reported for bamboos (19), and therefore, species-specific detection techniques are needed to observe A. take in host tissues. A number of staining techniques utilizing lectins and other chemical compounds have been used to observe naturally growing hyphae in host tissues (13). However, these approaches cannot be used to distinguish particular species. Therefore, such staining techniques using lectins and other chemical compounds are effective to differentiate fungi possessing particular morphological characteristics, for example, arbuscules, appressoria, or haustoria (33). Unfortunately, fungal endophytes are difficult to identify in host tissues, as discussed previously by Schulz and Boyle (24), partly because the morphologies of endophytic hyphae are usually different from those of cultured hyphae. To differentiate specific fungal species from other fungi, green fluorescent protein or related reporter protein transformants can be used (18, 36). This approach requires the development of genetic modification techniques for individual fungal species. In addition, this method cannot be applied to natural samples because it is necessary to inoculate host plants with fungal transformants. Although immunostaining techniques are also an effective approach (7, 26), they require complicated preparations for generating individual species-specific antibodies.In situ hybridization (ISH) with a species-specific probe is a potentially powerful technique for directly detecting endophytic fungi in natural samples. Species-specific rRNA-targeted oligonucleotide probes have been developed to detect and identify microorganisms in the environment (1, 6). ISH has also been used to detect fungal pathogens in animal tissues (10, 12, 14, 17). Despite these uses, only a few studies have applied this technique for the detection of fungal endophytes or epiphytes (16, 20). This is partly because the detection of fluorescence-labeled probes was hampered by the strong autofluorescence from the plant cell walls (23). However, Pirttilä et al. (20) succeeded in detecting fungal endophytes in meristematic tissues of Scots pine buds by the colorimetric ISH technique. In this study, ISH methodology using a 18S rRNA-targeted oligonucleotide probe was applied to visualize the location of the endophytic fungus A. take in bamboo with witches’ broom disease.     

A potential role for an extracellular methanol oxidase secreted by Moniliophthora perniciosa in Witches’ broom disease in cacao

The hemibiotrophic basidiomycete fungus Moniliophthora perniciosa, the causal agent of Witches’ broom disease (WBD) in cacao, is able to grow on methanol as the sole carbon source. In plants, one of the main sources of methanol is the pectin present in the structure of cell walls. Pectin is composed of highly methylesterified chains of galacturonic acid. The hydrolysis between the methyl radicals and galacturonic acid in esterified pectin, mediated by a pectin methylesterase (PME), releases methanol, which may be decomposed by a methanol oxidase (MOX). The analysis of the M. pernciosa genome revealed putative mox and pme genes. Real-time quantitative RT-PCR performed with RNA from mycelia grown in the presence of methanol or pectin as the sole carbon source and with RNA from infected cacao seedlings in different stages of the progression of WBD indicate that the two genes are coregulated, suggesting that the fungus may be metabolizing the methanol released from pectin. Moreover, immunolocalization of homogalacturonan, the main pectic domain that constitutes the primary cell wall matrix, shows a reduction in the level of pectin methyl esterification in infected cacao seedlings. Although MOX has been classically classified as a peroxisomal enzyme, M. perniciosa presents an extracellular methanol oxidase. Its activity was detected in the fungus culture supernatants, and mass spectrometry analysis indicated the presence of this enzyme in the fungus secretome. Because M. pernciosa possesses all genes classically related to methanol metabolism, we propose a peroxisome-independent model for the utilization of methanol by this fungus, which begins with the extracellular oxidation of methanol derived from the demethylation of pectin and finishes in the cytosol.     

In Vitro Production of Biotrophic-Like Cultures of Crinipellis perniciosa, the Causal Agent of Witches’ Broom Disease of Theobroma cacao

Witches’ broom disease (WBD) of cacao, caused by the hemibiotrophic fungus, Crinipellis perniciosa, exhibits a succession of symptoms that are caused by the biotrophic phase of the fungus. However, the study of this biotrophic phase is limited by its exclusive growth inside the plant or in the presence of callus. Here we report for the first time a method for the growth and maintenance of the biotrophic-like phase of C. perniciosa on a defined medium with metabolites found in the diseased tissues. Our results suggest that glycerol is a key carbon source for this interaction. This is a crucial achievement toward understanding the biology of this fungus during the infectious phase of WBD.     

Production of Calcium Oxalate Crystals by the Basidiomycete Moniliophthora perniciosa, the Causal Agent of Witches’ Broom Disease of Cacao

Oxalic acid has been shown as a virulence factor for some phytopathogenic fungi, removing calcium from pectin and favoring plant cell wall degradation. Recently, it was published that calcium oxalate accumulates in infected cacao tissues during the progression of Witches’ Broom disease (WBD). In the present work we report that the hemibiotrophic basidiomycete Moniliophthora perniciosa, the causal agent of WBD, produces calcium oxalate crystals. These crystals were initially observed by polarized light microscopy of hyphae growing on a glass slide, apparently being secreted from the cells. The analysis was refined by Scanning electron microscopy and the compositon of the crystals was confirmed by energy-dispersive x-ray spectrometry. The production of oxalate by M. perniciosa was reinforced by the identification of a putative gene coding for oxaloacetate acetylhydrolase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate. This gene was shown to be expressed in the biotrophic-like mycelia, which in planta occupy the intercellular middle-lamella space, a region filled with pectin. Taken together, our results suggest that oxalate production by M. perniciosa may play a role in the WBD pathogenesis mechanism.     

The Activity of TcCYS4 Modified by Variations in pH and Temperature Can Affect Symptoms of Witches’ Broom Disease of Cocoa,Caused by the Fungus Moniliophthora perniciosa

The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches’ broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches’ broom disease in cocoa.