Microsatellite analysis of Icelandic populations of the poplar fungal pathogen Melampsora larici-populina shows evidence of repeated colonization events

The basidiomycete Melampsora larici-populina causes foliar rust on Populus species from the sections Aigeiros and Tacamahaca, causing reduction in biomass production and economic losses. In the present study, samples of Icelandic M. larici-populina were collected for analysis of genetic diversity and population structure. A total of 439 isolates, collected at 15 locations, and analysed using 22 microsatellite markers were compared to data from French M. larici-populina populations. Twenty-one of the loci analysed were polymorphic, with an average of 3.4 alleles per locus. The mean observed and expected heterozygosities for all populations were 0.35 and 0.38. Evidence was found for a substructure within the Icelandic population with three subpopulations being the most likely scenario with low levels of gene flow. The population structure seen here is most likely shaped by both isolation and genetic drift as well as repeated events of colonization. In the future it can therefore be expected that regional poplar rust genotypes in Iceland change by two different modes; on one hand by transport of spores within the country and on the other hand by repeated colonization events. The results reported here underline the importance of closely monitoring the development of fungal diseases in Iceland, and to carefully select for resistance in Icelandic plant breeding programs.     

Cloning of <Emphasis Type="Bold">β</Emphasis>-tubulin and succinate dehydrogenase genes from <Emphasis Type="Italic">Uromyces fabae</Emphasis> and establishing selection conditions for their use in transformation

The obligate biotrophic nature of rust fungi calls for an in planta selection scheme as a means of developing a rust transformation technology. We show that the fungicides benomyl (used as its formulated product benlate) and carboxin suppress morphogenesis of the rust fungus Uromyces fabae in vitro and disease in planta, the latter without affecting the health of the host. The limits of their applicability were determined regarding concentration, method of application and optimal time intervals of treatment. Besides procedures for selection, a stable transformation system will also need to include genetic markers allowing to enrich for transformed cells within a large background of untransformed cells. Since the molecular targets of benlate and carboxin had been identified as -tubulin and succinate dehydrogenase, respectively, the corresponding genes (Uf-TBBIand Uf-SUCDHI) were cloned and characterized. Molecular phylogenies demonstrate that both are typical homologs to those of other Basidiomycota. RT-PCR analysis confirmed that both genes are constitutively expressed in all developmental stages of the mitotic uredospore multiplication cycle. Since homologs of Uf-TBB1and Uf-SUCDH1 have been successfully used as selection markers in other fungal systems, they provide valuable tools to develop additional corner stones of a stable transformation system for rust fungi.     

小豆锈病病原菌鉴定

 小豆锈病是小豆的重要病害。然而,小豆锈病病原菌在我国还没有被鉴定。本文对从黑龙江省和内蒙古自治区采集的5个小豆锈病菌样品的分类地位进行了研究。形态学观察表明,小豆锈病菌夏孢子芽孔位于赤道线或赤道线上方,冬孢子平均壁厚为2.0 μm。分子标记检测发现,小豆锈病菌不能被疣顶单胞锈菌特异性引物对UA-ITSF/UA-ITSR扩增,小豆锈病菌和豇豆锈病菌不能被豇豆单胞锈特异性引物对UV-ITSF/UV-ITSR区分。对豇豆单胞锈特异性引物对扩增的4个小豆锈病菌样品PCR产物进行测序,比对分析表明目标序列与小豆单胞锈ITS序列的同源性为99%~100%。基于夏孢子的芽孔位置、冬孢子壁的厚度、疣顶单胞锈菌及豇豆单胞锈特异性引物检测结果和ITS序列分析,小豆锈病菌被鉴定为小豆单胞锈。     

The haustorial host-parasite interface in rust-infected bean leaves after high-pressure freezing

The haustorial fine structure of the bean rust fungus, Uromyces appendiculatus var. appendiculatus, was studied within the cells of its host, Phaseolus vulgaris. Results were obtained after high-pressure freezing and subsequent freeze-substitution or freeze-fracturing. Good preservation of leaf tissue after freeze-substitution needed cryoprotection with 8% methanol. For freeze-fracturing, no chemical treatment was applied. In addition to the organelles which are generally found in fungi after cryo-fixation, tubular-vesicular complexes were found in the cytoplasm. Both techniques revealed an extrahaustorial matrix of even width, surrounding the haustorial body. The extrahaustorial membrane was not undulated, and the side facing the plant cytoplasm was lined with a delicate fringe of well-stained material. The extrahaustorial membrane was nearly devoid of intramembrane particles. The host plasma membrane in infected tissue, especially the protoplasmic face, had fewer intramembrane particles than those in uninfected tissue. The haustorial plasma membrane contained many intramembrane particles.     

Melampsora larici-populina, the main rust pathogen, causes loss in biomass production of black cottonwood plantations in the south of China

Epidemic outbreaks of rust disease have been observed in most of the black cottonwood (Populus trichocarpa) plantations in the south of China in recent years. However, the exact pathogens that cause rust disease in this area remain largely unknown. In this study we collected rust fungi from black cottonwood plantations at four different places in the south of China. Examination of these fungi by scanning electron microscope (SEM) and light microscopy (LM) revealed that the morphological characteristics of urediniospores for all the collected fungi were very close to that of Melampsora larici-populina. Using species-specific primers, these pathogens were confirmed to be M. larici-populina. In a survey on the resistance to rust disease for 88 genotypes of black cottonwood, nine potential candidate genotypes were detected that may be free from infection of rust pathogen. The results of this study provide essential information for breeding new rust-resistant black cottonwood cultivars.     

Fungitoxicity of bromo- and iodo-3,5-dinitrobenzoates

Esters of 2-bromo-, 4-bromo-, 2-iodo- and 4-iodo- 3,5-dinitrobenzoic acids show considerable antifungal activity in spore germination tests against Alternaria brassicicola, Botrytis cinerea, Septoria nodorum and Uromyces fabae. They act as protectant fungicides against Erysiphe graminis, Puccinia recondita and Septoria nodorum on wheat and Botrytis fabae on broad bean. The most active compounds are methyl and ethyl 4-bromo-3,5-dinitrobenzoates which give better control of wheat rust than oxycarboxin and methyl 4-iodo-3,5-dinitrobenzoate which is at least as effective as captan against Septoria leaf spot on wheat. Several of the esters are superior to captan in controlling chocolate spot on broad bean.     

Effect of flutianil on the morphology and gene expression of powdery mildew

Flutianil, a fungicide effective only on powdery mildew, was previously reported to affect the host cell''s haustorial formation and nutrient absorption. Studies were conducted to investigate flutianil''s primary site of action on Blumeria graminis morphology using transmission electron microscope (TEM) observation and RNA sequencing (RAN-seq) techniques. TEM observation revealed that flutianil caused the extra-haustorial matrix and fungal cell wall to be obscured, without remarkable changes of other fungal organelles. RNA-seq analysis indicated that, unlike other powdery-mildew fungicides, flutianil did not significantly affect the constantly expressed genes for the survival of B. graminis. Genes whose expression is up- or downregulated by flutianil were found; these are the three sugar transporter genes and various effector genes, mainly expressed in haustoria. These findings indicate that the primary site of action of flutianil might be in the haustoria.     

The Effect of Nitrogen on Disease Development and Gene Expression in Bacterial and Fungal Plant Pathogens

Successful colonisation of plants by pathogens requires efficient utilisation of nutrient resources available in host tissues. Several bacterial and fungal genes are specifically induced during pathogenesis and under nitrogen-limiting conditions in vitro. This suggests that a nitrogen-limiting environment may be one of the cues for disease symptom development during growth of the pathogens in planta. Here we review recent literature on the effect of nitrogen and nitrogen-regulated genes on disease development, caused by phytopathogenic bacteria and fungi. Furthermore, the potential influence of nitrogen-limitation or general nutrient limitation on several in planta-induced bacterial and fungal pathogenicity, virulence and avirulence genes will be discussed.     

Powdery Mildew of Prairie Gentian: Characteristics,Molecular Phylogeny and Pathogenicity

In March 1999, we found prairie gentian (Eustoma grandiflorum) infected with powdery mildew in a greenhouse in Oita Prefecture, Japan. Morphological observation revealed that the causal fungus belongs to the mitosporic genus Oidium subgenus Pseudoidium [teleomorph: Erysiphe sensu Braun and Takamatsu (2000)]. Precise taxonomic position of the fungus, however, is uncertain due to lack of the perfect stage. We determined the nucleotide sequence of the rDNA ITS region of the fungus. Comparison of the sequence with those obtained from DNA databases of this fungal group revealed that the sequence is identical to those of powdery mildews from garden four-o'clock (Mirabilis jalapa) and broad bean (Vicia faba). Inoculation of an isolate from garden four-o'clock caused mildew on prairie gentian and broad bean, suggesting that the prairie gentian mildew originates from garden four-o'clock or broad bean. Molecular phylogenetic analysis indicated a close relationship of this fungus to Erysiphe baeumleri on Vicia spp. and E. trifolii on Trifolium pratense. From these results, we propose that prairie gentian mildew diverged from a Fabaceae-parasitic ancestor. Received 14 March 2002/ Accepted in revised form 28 May 2002     

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

河南商丘地区棉花黄萎病菌分离鉴定和致病力分析

为探讨河南商丘地区棉花黄萎病菌的致病型群体变异,对该地区棉花上分离的8株单孢菌株的菌落形态、显微结构、致病力、ITS序列、系统进化及菌体蛋白等方面进行了研究。结果表明:这些菌株均属于棉花黄萎病菌Verticilliumdahliae;系统进化树显示8株菌株并没有聚在同一进化枝上;8株黄萎菌菌株存在致病力差异,SQ4菌株致病力最强,属于落叶型,而其它致病力较弱的7个菌株属于非落叶型;不同致病力的菌株间蛋白谱带存在差异。     

The use of host resistance in disease management of rust in common bean

Bean rust, caused by Uramyces appendiculatus, is one of the major diseases in dry and snap bean production world-wide. Numerous advancements in disease management have been made to reduce rust losses. Host resistance is an important component of rust management. However, durability of disease resistance has often been short due to the use of single genes for resistance interacting with extremely high virulence diversity of the bean rust fungus. The challenge to increase durability of resistance has led to strategies such as gene pyramiding of race-specific resistance, selection and use of partial resistance, and investigation and discovery of leaf morphological features that may slow the rust epidemic. Germplasm with multiple sources of rust resistance has been developed in specific bean seed classes and released for public and commercial use in intensive production systems such as those in the United States. However, progress to develop rust resistant germplasm for the subsistence agriculture of Latin America and Africa where intercropping and mixed cultivars dominate the production system has been slow. Incorporation of high yielding, disease-resistant components as partial replacement in farmer's mixtures has the potential to reduce severity in the crop and increase yield in the presence of rust. This strategy would not erode the genetic diversity that is historically known to enhance resistance durability and for many years has given stability in production in the subsistent agriculture systems.     

Histological studies of the expression of the Lr9, Lr20 and Lr28 alleles for resistance to leaf rust in wheat

The development of the leaf rust fungus ( Puccinia recondita f.sp. tritici ) in a susceptible cultivar and three other cultivars possessing the Lr9, Lr20 and Lr28 alleles for resistance was studied by light and fluorescence microscopy. Formation of the substomatal vesicle, intercellular hypha and the first haustorial mother cell was unaffected by resistance. Lr9 and Lr28 expression was rapid, first seen as early initiation of hyphal branching at 16 h after inoculation, then reduced haustorial diameters at 19 h. Limited host cell necrosis was seen immediately afterwards. Elongation of intercellular hyphae was reduced between 20 and 24 h, and virtually ceased by about 30 h. Numbers of infection sites with a second haustorial mother cell were briefly higher at 24 h. Reduced hyphal branching and haustorial mother cell numbers were seen at 20–24 h and 36 h respectively. Lr20 expression was not seen until 36 h when reduced hyphal branching was observed, accompanied by extensive host cell necrosis. Reduced haustorial mother cell numbers were detected at 48 h. Findings suggested a secondary role for host cell necrosis in the expression of the Lr9 and Lr28 alleles. Host necrosis may play a determinant role in Lr20- based expression.