Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.

Late blight of potato caused by Phytophthora infestans is one of the most damaging diseases affecting potato production worldwide. We screened 357 root fungal endophytes isolated from four solanaceous plant species obtained from Kenya regarding their in vitro antagonistic activity against the potato late blight pathogen and evaluated their performance in planta. Preliminary in vitro tests revealed that 46 of these isolates showed potential activity against the pathogen. Based on their ITS-sequences, 37 out of 46 endophytes were identified to species level, three isolates were connected to higher taxa (phylum or genus), while two remained unidentified. Confrontation assays, as well as assays for volatile or diffusible organic compounds, resulted in the selection of three endophytes (KB1S1-4, KA2S1-42, and KB2S2-15) with a pronounced inhibitory activity against P. infestans. All three isolates produce volatile organic compounds that inhibit mycelial growth of P. infestans by up to 48.9%. The addition of 5% extracts obtained from KB2S2-15 or KA2S1-42 to P. infestans sporangia entirely suppressed their germination. A slightly lower inhibition (69%) was achieved using extract from KB1S1-4. Moreover, late blight symptoms and the mycelial growth of P. infestans were completely suppressed when leaflets were pre-treated with a 5% extract from these endophytes. This might suggest the implementation of such biocontrol candidates or their fungicidal compounds in late blight control strategies.

Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53–85% and 61–87%, respectively, and reduced TRI5 expression by 84–97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.

Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended.

Uromyces fabae is a major pathogen of broad bean, Vicia faba. U. fabae has served as a model among rust fungi to elucidate the development of infection structures, expression and secretion of cell wall degrading enzymes and gene expression. Using U. fabae, enormous progress was made regarding nutrient uptake and metabolism and in the search for secreted proteins and effectors. Here, we present results from a genome survey of U. fabae. Paired end Illumina sequencing provided 53 Gb of data. An assembly gave 59,735 scaffolds with a total length of 216 Mb. K-mer analysis estimated the genome size to be 329 Mb. Of a representative set of 23,153 predicted proteins we could annotate 10,209, and predict 599 secreted proteins. Clustering of the protein set indicates families of highly likely effectors. We also found new homologs of RTP1p, a prototype rust effector. The U. fabae genome will be an important resource for comparative analyses with U. appendiculatus and P. pachyrhizi and provide information regarding the phylogenetic relationship of the genus Uromyces with respect to other rust fungi already sequenced, namely Puccinia graminis f. sp. tritici, P. striiformis f. sp. tritici, Melampsora lini, and Melampsora larici-populina.

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.