Identification of genes expressed by Phakopsora pachyrhizi,the pathogen causing soybean rust,at a late stage of infection of susceptible soybean leaves

Soybean is one of the top five agricultural products in the United States and is highly susceptible to Phakopsora pachyrhizi, an exotic obligate biotrophic fungus. The little amount of genomic information about P. pachyrhizi limits understanding of the soybean–soybean rust pathogen interaction and the possibility of engineering resistance to this pathogen in soybean. Illumina mRNA‐Seq analysis revealed P. pachyrhizi genes expressed during a biotrophic interaction between P. pachyrhizi and soybean during fungal sporulation 10 days after inoculation. Approximately 2·4 million DNA sequences representing portions of potential P. pachyrhizi genes were assembled into 32 940 contigs that were used to search against expressed sequence tag (EST), protein and conserved domain databases. About 7500 contigs represent newly discovered P. pachyrhizi sequences. Of these, 527 shared similarity to genes encoding fungal proteins involved in different metabolic pathways such as galactose and glycogen metabolism, glycolysis, the citrate cycle, fatty acid metabolism, amino acid metabolism, proteolysis, protein synthesis, cell cycle division and mitosis, and cell wall biogenesis. Almost 7000 potential P. pachyrhizi genes are still of unknown function. Such information may be useful in the development of new methods of broadening resistance of soybean to P. pachyrhizi, including the silencing of important P. pachyrhizi genes, and also to understand the molecular basis of soybean–P. pachyrhizi interactions.     

Evaluation of soybean genotypes for resistance against the rust-causing fungus Phakopsora pachyrhizi in East Africa

Soybean rust, caused by the biotrophic fungus Phakopsora pachyrhizi, is the most important foliar disease of soybean (Glycine max) worldwide. Deployment of resistant soybean cultivars is the best option for managing this disease. Genes conferring resistance to P. pachyrhizi have been identified, but pathotypes of the rust fungus overcoming these resistance genes have also been found. To identify novel resistance genes, soybean genotypes from both local and international sources were screened at multiple locations in Tanzania and Uganda in 2016 and 2017. The results from this screening revealed that infection types, disease severities, and sporulation levels varied among the genotypes and locations. The majority of the genotypes had tan-coloured (TAN) lesions and developed moderate sporulation, implying susceptibility, while only seven of the 71 lines had reddish-brown (RB) lesions and showed low disease severities in all of the screening environments. We identified seven genotypes that were the most resistant to rust in the most locations over the two years. These genotypes will be useful for further studies and, ultimately, for rust management, as they show broad resistance to various pathotypes of the rust fungus.     

Expressed sequence tags from the phytopathogenic fungus Botrytis cinerea

A large set of genes was identified in the phytopathogenic fungus Botrytis cinerea by using an expressed sequence tag approach. The fungus was grown in axenic culture and a cDNA library was produced. From this library, 6559 ESTs were obtained. The combined sequences represent 3026 unisequences that corresponds to approximately one-quarter of the estimated total number of genes in B. cinerea. Approximately 18% of the ESTs showed significant similarities with genes coding for proteins with known functions,~56% were similar to genes coding for proteins with unknown functions and ~26% were orphans. A substantial B. cinerea gene inventory including putative virulence factors was therefore obtained and is now available at the Génoplante-Info Database interface (http://urgi.infobiogen.fr///Projects/GPiDB/Interface/).     

Movement of <Emphasis Type="Italic">Phakopsora pachyrhizi</Emphasis> (soybean rust) spores by non-conventional means

Soybean, caused by the rust fungus Phakopsora pachyrhizi, is the most important foliar pathogen infecting soybean. Historically, the disease was important only in the Eastern Hemisphere, but since 1994 the disease has been reported in many countries in Africa and the Americas. In the U.S.A., soybean rust has been perceived as a threat to soybean production and monitoring of the disease occurs throughout the country where soybean is grown. The objectives of this study were to show conclusive evidence that soybean rust spores can be transported by non-conventional means such as clothing. The implication may affect how researchers approach monitoring this disease in research and sentinel plots.     

The origin and genetic diversity of the causal agent of Asian soybean rust,Phakopsora pachyrhizi,in South America

A sequence‐based approach was used to investigate molecular genetic variations in Phakopsora pachyrhizi, an obligate biotrophic pathogen that causes Asian soybean rust. In Argentina, the samples came from uredinium‐bearing leaves taken from 11 soybean fields; in Brazil, the samples comprised urediniospores from leaves of 10 soybean genotypes that had been grown in three experimental stations during two growing seasons. PCR‐based cloning techniques were used to generate DNA sequences for two gene regions and alignments were supplemented with data from GenBank. A total of 575 sequences for the internal transcribed spacer region (18 ribotypes) and 160 partial sequences for a housekeeping gene encoding ADP‐ribosylation factor (10 haplotypes) were obtained. Ribotype accumulation curves predicted that about 20 bacterial clones would recover 5–6 ribotypes (c. 70–80% of the total molecular variation) per locality. The samples from the three experimental stations in Brazil displayed most (14 out of 16) ribotypes found worldwide; the lack of genetic structure and differentiation at a diverse geographic scale suggests that both local and distant sources provide airborne inoculum during disease establishment. Soybean genotypes with resistance genes for the Asian soybean rust did not decrease the molecular genetic variation of fungal populations.     

Soybean production in eastern and southern Africa and threat of yield loss due to soybean rust caused by Phakopsora pachyrhizi

Soybean is a major source of oil and proteins worldwide. The demand for soybean has increased in Africa, driven by the growing feed industry for poultry, aquaculture and home consumption in the form of processed milk, baked beans and for blending with maize and wheat flour. Soybean, in addition to being a major source of cooking oil, is also used in other industrial processes such as in the production of paints and candle wax. The demand for soybean in Africa so far outweighs the supply, hence the deficit is mainly covered through imports of soybean products such as soybean meal. The area under soybean production has increased in response to the growing demand, a trend that is expected to continue in the coming years. As the production area increases, diseases and insect pests, declining soil fertility and other abiotic factors pose a major challenge. Soybean rust disease, caused by the fungus Phakopsora pachyrhizi, presents one of the major threats to soybean production in Africa due to its rapid spread as a result of the ease by which its spores are dispersed by the wind. Disease control by introducing resistant soybean varieties has been difficult due to the presence of different populations of the fungus that vary in pathogenicity, virulence and genetic composition. Improved understanding of the dynamics of rust ecology, epidemiology and population genetics will enhance the effectiveness of targeted interventions that, in turn, will safeguard soybean productivity.     

Pathogenic diversity of soybean rust in Argentina, Brazil, and Paraguay

Phakopsora pachyrhizi, the cause of soybean rust, is an economically important pathogen of soybean in South America. Understanding the pathogenicity of indigenous fungal populations is useful for identifying resistant plant genotypes and targeting effective cultivars against certain populations. Fifty-nine rust populations from Argentina, Brazil, and Paraguay were evaluated for pathogenicity in three cropping seasons, 2007/2008–2009/2010, using 16 soybean differentials. Only two pairs of P. pachyrhizi populations displayed identical pathogenicity profiles, indicating substantial pathogenic variation in the rust populations. Comparative analysis of 59 South American and five Japanese samples revealed that pathogenic differences were not only detected within South America but also distinct between the P. pachyrhizi populations from South America and Japan. In addition, seasonal changes in rust pathogenicity were detected during the sampling period. The differentials containing resistance genes (Rpp: resistance to P. p achyrhizi) Rpp1, Rpp2, Rpp3, and Rpp4, except for Plant Introduction (PI) 587880A, displayed a resistant reaction to only 1.8–14, 24–28, 22, and 36 % of South American P. pachyrhizi populations, respectively. In contrast, PI 587880A (Rpp1), Shiranui (Rpp5), and 3 Rpp-unknown differentials (PI 587855, PI 587905, and PI 594767A) showed a resistant reaction to 78–96 % of all populations. This study demonstrated that P. pachyrhizi populations from South America vary geographically and temporally in pathogenicity and that the known Rpp genes other than Rpp1 in PI 587880A and Rpp5 have been less effective against recent pathogen populations in the countries studied.     

A major variation in the virulence of the Asian soybean rust pathogen (Phakopsora pachyrhizi) in Bangladesh

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a major threat to soybean production in Bangladesh. Understanding the yearly changes and the current status of pathogenic structures is essential for developing appropriate breeding strategies for obtaining ASR-resistant soybean lines. Thirty-four P. pachyrhizi samples were collected from ASR hotspot areas (Chandpur, Lakshmipur, Noakhali, Barisal and Bhola districts) of Bangladesh in 2018 and 2019 and evaluated for pathogenicity on 12 soybean differential lines. The tested samples showed similar and dissimilar pathogenicity patterns on the differentials, yielding 21 distinct pathotypes. The cluster analysis, principal coordinate analysis and principal component analysis of the disease phenotypes of 47 samples collected in 2016, 2018 and 2019 indicated a higher pathogenic diversity and virulence variation in the P. pachyrhizi samples of 2018 and 2019 compared to that of 2016. The pathogenicity profiles of the Bangladeshi P. pachyrhizi samples appeared distinct from those of Argentinian and Brazilian samples, but showed slight similarities with Japanese, Mexican and Paraguayan samples. Furthermore, none of the resistance genes for P. pachyrhizi (Rpp genes) was solely effective against all the tested samples from 2018 and 2019, while samples (BdRP-48, BdRP-56 and BdRP-58) virulent to all Rpp1–Rpp6 genes were detected. The Rpp-pyramided line No6–12–1, carrying Rpp2, Rpp4 and Rpp5, was capable of conferring robust resistance to these virulent samples. Altogether, these results indicate an increase in the virulence of the current ASR pathogen in Bangladesh, which can be resolved by pyramiding different resistance genes in soybean cultivars.     

Subcelullar localization of proteins associated with <Emphasis Type="Italic">Prune dwarf virus</Emphasis> replication

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose, one of the most severe diseases of the common bean (Phaseolus vulgaris). The infection process begins with the adhesion of conidia to the plant’s surface. Appressoria are then formed, allowing penetration of the fungus. Next, the biotrophic phase begins, followed by the necrotrophic phase. Due to the peculiar nutrition mode of the fungus, including both of the previously mentioned stages, it is of great interest to determine which genes are involved in the transition between the two phases during the infection process. To determine this, suppression subtractive hybridization (SSH) was used in association with qRT-PCR in the present study. These methods allowed for the identification of genes that were differentially expressed at each developmental stage of the fungus in the plant. This is the first report on the use of the cited techniques to evaluate the infectious cycle of the fungus. A total of 175 sequences exhibited significant identity (e?≤?10^?5) with sequences present in the sequenced genomes of P. vulgaris and C. lindemuthianum; approximately 41 % of those were determined to belong to the fungus, and 59 % were determined to belong to the plant. Of the predicted sequences, 68 % were of unknown function or were not found in the databases. Among the analyzed expressed sequence tags (ESTs), sequences were found that encode proteins related to: primary and secondary metabolism; the transport of different compounds; the degradation/modification of proteins; cell regulation and signaling; cellular stress response; and the degradation of exogenous compounds. The obtained results allowed for the identification of sequences encoding proteins that are essential for the progression of anthracnose. Furthermore, it was possible to identify new genes, the functions of which have not yet been described, and even to identify unique genes of C. lindemuthianum that are involved in the pathogenicity and virulence of this fungus.     

Nickel potentiates soybean resistance against infection by Phakopsora pachyrhizi

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, causes significant yield losses worldwide. Nickel (Ni) plays a key role in the metabolism of some profitable crops, such as soybeans, because it is a constituent of several biomolecules and is required for the catalytic process of several enzymes. This study investigated the effect of foliar Ni treatment on the potentiation of soybean cultivar TMG 135 resistance to P. pachyrhizi infection at the microscopic, biochemical, and molecular levels. The severity of ASR decreased by 35% in plants treated with Ni. The malondialdehyde concentration, an indicator of cellular oxidative damage, was high in the leaves of plants that were not treated with Ni and was linked to ASR severity and the extensive colonization of the palisade and spongy parenchyma cells by fungal hyphae. The lignin concentration, β-1,3-glucanase activity, and expression of the URE gene and the defence-related genes PAL1.1, PAL2.1, CHI1B1, and PR-1A were up-regulated in Ni-treated plants infected with P. pachyrhizi. The information provided by this study shows the great potential of Ni to increase the basal level of soybean resistance to ASR and to complement other control methods within the context of sustainable agriculture.     

Diversity and distribution of pathotypes of the soybean rust fungus Phakopsora pachyrhizi in East Africa

Phakopsora pachyrhizi is a biotrophic fungus that causes rust on soybean, leading to devastating yield losses. Development of resistant cultivars for deployment in different geographic regions requires a comprehensive understanding of the prevalent P. pachyrhizi pathotypes. To determine the pathotypes existing in four East African countries, 65 isolates were tested on 11 soybean host differentials. In addition, the virulence spectrum of isolates collected from the same region over multiple years was compared. The majority of the isolates (54%) belonged to pathotype 1000, which was found in all countries. The pathotypes with the most complex virulence spectrum, which comprised isolates from Kenya and Malawi, were virulent on four differentials. All pathotypes were virulent on soybean genotypes carrying the Rpp1 resistance gene to P. pachyrhizi, but they were avirulent on cultivars carrying the Rpp1b, Rpp2, or Rpp3 gene, as well as on cultivar No6-12-1 that carries Rpp2, Rpp4, and Rpp5. Two of the pathotypes were virulent on cultivar UG 5 that carries Rpp1 and Rpp3 and on Hyuuga that carries Rpp3 and Rpp5. The isolates collected from different countries differed in their virulence spectrum across the years. Shannon's index (H) and Simpson's index (S) of diversity indicated that the isolates from Malawi were more diverse (H = 1.55, S = 0.90) while those from Uganda had lower diversity (H = 0.78, S = 0.46 ). The Rpp genes that were found to provide resistance to all pathotypes of P. pachyrhizi can be employed for soybean breeding aimed at durable rust resistance.     

Essential oils for preventative treatment and control of Asian soybean rust

The objective of the work was to evaluate the effect of essential oils as a preventive treatment to control Asian soybean rust. Initially the fungitoxic effect of the essential oils of Hyptis marrubioides, Aloysia gratissima and Cordia verbenacea was tested on the urediniospores of Phakopsora pachyrhizi, through in vitro tests. The in vivo test was set up in a greenhouse, using cultivar MGBR-46. The treatments consisted of the three oils at different concentrations, a fungicide based on pyraclostrobin + epoxyconazole and a control. To verify the potential of the essential oils in preventive treatment, inoculation was conducted at 0, 6, 12 and 24?h intervals, after application of the treatments. All treatments inhibited 100% of Phakopsora pachyrhizi germination. In the in vivo test, it was observed that all of the oils were efficient in controlling soybean rust, by preventive treatment, mainly at the higher concentrations. Efficiency of the oils was reduced with the increase of the interval between the application of the treatments and the onset of the pathogen. The essential oils, at the tested dosages, were not as efficient as the pyraclostrobin + epoxyconazole based fungicide.     

Recent outbreaks of rust diseases and the importance of basic biological research for controlling rusts

Rust fungi are obligate plant parasites belonging to the order Pucciniales; they comprise about 7,800 species throughout the world. Some species seriously damage crops, vegetables, fruits and trees. Of these species, wheat stem rust (Puccinia graminis f. sp. tritici), Asian soybean rust (Phakopsora pachyrhizi) and myrtle rust (Puccinia psidii) have recently become major concerns worldwide, and this review, discusses recent rust disease outbreaks of Asian soybean rust and myrtle rust. Both rusts have very wide host ranges. Asian soybean rust has spread from its original region of distribution (eastern Asia) to many areas of soybean cultivation around the world. Myrtle rust is a new disease in areas where host plants were first introduced and has spread to other parts of the world including the areas where the host plants are indigenous. New diseases of economically important plants can occur by host shifts from wild host plants or host jumps from phylogenetically unrelated plant species. Recent advances in molecular phylogenetic studies have contributed to a revision of rust taxonomy. Molecular phylogenetic analyses, together with precise morphological observations and inoculation experiments, have identified taxonomic groups among populations that are morphologically very similar. Systematic, ecological and other basic biological studies of rust fungi in both cultivated and wild host plants are very important for developing methods to control rust diseases. Recent changes in the International Code of Botanical Nomenclature will surely affect the systematics of rust fungi.     

小麦条锈菌cDNA文库构建和表达序列标签(ESTs)分析

 小麦条锈菌(Puccinia striiformis f. sp. tritici)是全世界范围内小麦生产上的重要病原真菌, 但是对小麦条锈菌的基因组和基因功能却了解甚少。为了促进小麦条锈菌基因组学的发展和大规模基因发现, 我们以噬菌体λTrip1Ex2为载体, 采用SMART技术构建了小麦条锈菌萌发夏孢子的cDNA文库。原始文库的滴度为1.1×10⁶pfu/mL, 平均插入片段长度为750 bp。从文库中随机挑取279个cDNA克隆测序, 分析发现这些ESTs的平均GC含量为45.08%。通过聚类分析, 279个ESTs拼接成31个contigs和80 singletons。BLASTx分析表明, 47%的ESTs与GenBank中报道的功能已知或未知蛋白具有相似性。tBLASTx分析表明12个uniseqs与EST数据库中的序列具有相似性, 其中9个是来自担子菌的cDNA文库。几个EST与已知的真菌致病相关基因具有高度相似性。RT-PCR分析了几个基因在小麦条锈菌侵染过程中的表达水平。这些结果为小麦条锈菌夏孢子萌发以及侵染寄主过程中的基因表达研究奠定了很好的分子生物学基础。     

Molecular mapping of Asian soybean rust resistance in soybean landraces PI 594767A,PI 587905 and PI 416764

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most serious diseases of soybean. The soybean landraces PI 594767A, PI 587905 and PI 416764 previously showed high levels of resistance to a wide range of ASR fungus, while the genetic basis of the resistance has yet to be understood. In this study, the ASR resistance loci were mapped using three independent mapping populations, POP‐1, POP‐2 and POP‐3 derived from crosses BRS184 × PI 594767A, BRS184 ×  PI 587905 and BRS184 × PI 416764, respectively. In each population, the resistance to ASR segregated as a single gene, but the resistance was dominant in PI 594767A and PI 587905 and incompletely dominant in PI 416764. The resistance genes from both PI 594767A and PI 587905 were mapped on chromosome 18 corresponding to the same location as known resistance locus Rpp1. Quantitative trait locus (QTL) analysis performed on POP‐3 identified the putative ASR resistance locus in PI 416764 on the defined region of chromosome 6 where Rpp3 was located. The QTLs detected by the mapping explained about 67–72% of the phenotypic variation in POP‐3. Cluster analysis based on disease reactions to 64 ASR populations demonstrated the presence of at least two types of functional resistant Rpp1 alleles: strong and weak allele(s), e.g. soybean accession PI 594767A and PI 587905 carry the strong resistant Rpp1 allele(s). Introducing or pyramiding strong Rpp1 allele(s) in elite soybean cultivars is expected to be useful against the South American rust population.     

Amino acid substitutions responsible for different QoI and SDHI sensitivity patterns in Puccinia horiana,the causal agent of chrysanthemum white rust

Quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are major groups of agricultural fungicides. However, resistance to some of these fungicides has been reported in a Japanese population of Puccinia horiana, the causal agent of chrysanthemum white rust disease. Because their mechanisms are not well understood, we investigated the existence of mutations in QoI and SDHI target protein-encoding genes. Eight out of nine isolates from cultivated chrysanthemum carried L275F and L299F amino acid substitutions in cytochrome b, the target protein of QoIs. These isolates showed 23- and 17-fold higher EC₅₀ values for the QoI fungicides azoxystrobin and kresoxim-methyl, respectively, in basidiospore germination inhibitory tests, while they were hypersensitive to another QoI, famoxadone. All nine isolates were resistant to SDHI oxycarboxin and carried the I88F substitution in SdhC. This substitution was orthologous to the SdhC-I86F substitution found in some Brazilian isolates of the soybean rust fungus, Phakopsora pachyrhizi, showing reduced sensitivity to some SDHIs. Although the rarity of wild-type sensitive isolates, the subsequent limited number of comparisons between wild types and mutants, and a difficulty in applying reverse genetic analysis to this obligate parasite, are obstacles in making definitive conclusions, L275F and L299F in cytochrome b and SdhC-I88F are suspected to be responsible for the different patterns of sensitivity to QoI and for oxycarboxin-resistance in P. horiana, respectively.