Gene deletion and constitutive expression of the pectate lyase gene 1 (MoPL1) lead to diminished virulence of Magnaporthe oryzae.

Phytopathogenic fungi are known to secrete specific proteins which act as virulence factors and promote host colonization. Some of them are enzymes with plant cell wall degradation capability, like pectate lyases (Pls). In this work, we examined the involvement of Pls in the infection process of Magnaporthe oryzae, the causal agent of rice blast disease. From three Plgenes annotated in the M. oryzae genome, only transcripts of MoPL1 considerably accumulated during the infection process with a peak at 72 h post inoculation. Both, gene deletion and a constitutive expression of MoPL1 in M. oryzae led to a significant reduction in virulence. By contrast, mutants that constitutively expressed an enzymatic inactive version of MoPl1 did not differ in virulence compared to the wild type isolate. This indicates that the enzymatic activity of MoPl1 is responsible for diminished virulence, which is presumably due to degradation products recognized as danger associated molecular patterns (DAMPs), which strengthen the plant immune response. Microscopic analysis of infection sites pointed to an increased plant defense response. Additionally, MoPl1 tagged with mRFP, and not the enzymatic inactive version, focally accumulated in attacked plant cells beneath appressoria and at sites where fungal hyphae transverse from one to another cell. These findings shed new light on the role of pectate lyases during tissue colonization in the necrotrophic stage of M. oryzae's life cycle.

Colletotrichum spp. from Soybean Cause Disease on Lupin and Can Induce Plant Growth-Promoting Effects.

Protein crop plants such as soybean and lupin are attracting increasing attention because of their potential use as forage, green manure, or for the production of oil and protein for human consumption. Whereas soybean production only recently gained more importance in Germany and within the whole EU in frame of protein strategies, lupin production is already well-established in Germany. The cultivation of lupins is impeded by the hemibiotrophic ascomycete Colletotrichum lupini, the causal agent of anthracnose disease. Worldwide, soybean is also a host for a variety of Colletotrichum species, but so far, this seems to not be the case in Germany. Cross-virulence between lupin- and soybean-infecting isolates is a potential threat, especially considering the overlap of possible soybean and lupin growing areas in Germany. To address this question, we systematically investigated the interaction of different Colletotrichum species isolated from soybean in Brazil on German soybean and lupin plant cultivars. Conversely, we tested the interaction of a German field isolate of C. lupini with soybean. Under controlled conditions, Colletotrichum species from soybean and lupin were able to cross-infect the other host plant with varying degrees of virulence, thus underpinning the potential risk of increased anthracnose diseases in the future. Interestingly, we observed a pronounced plant growth-promoting effect for some host-pathogen combinations, which might open the route to the use of beneficial biological agents in lupin and soybean production.

First report of Boeremia exigua var. exigua causing Black Spot-like symptoms on commercially grown soybean in Germany.

Soybean (Glycine max [L.] Merr.) is economically the most important protein crop grown worldwide. However, Europe largely depends on soybean imported from the Americas (European Commission 2019; Haupt and Schmid 2020). In Germany, soybean production was not formally recorded before 2016, but since then a steady increase along with an expansion of the growing area from the south of Germany to northern states occurred. In 2019 an area of 29,000 hectares was under soybean cultivation (Federal Ministry of Food and Agriculture (Germany) 2019). In the state of North Rhine-Westphalia (NRW, western part of Germany) farmers have started in recent years to cultivate soybean, making it increasingly important to monitor pathogens associated with this new crop. At the beginning of October 2019, shortly before harvest, rows of black spots on pods and stems of soybean plants cv. Viola throughout a field site near Jülich (NRW) were observed. Close observation identified them as pycnidia with similarity to symptoms reported from soybean in Austria in 2015 (Hissek and Bedlan 2016). The collected samples were thoroughly surface sterilized (two washes with 70 % EtOH, a rinse in 0.5 % sodium hypochlorite solution and a final wash in sterile double distilled water) and placed on plates containing potato dextrose agar (PDA) at 22 °C in the dark. Fungal colonies were transferred to malt extract agar plates (MEA) and examined by microscopy. Thus, 34 of 41 isolates looked morphologically similar, producing colonies that appeared dark grey with white aerial mycelium and round to irregular margins. A single spore isolate was generated and designated IPP1903. Spores derived from IPP1903 were unicellular and mostly oblong to cylindrical with a mean width of 2.6±0.3 µm and a mean length of 5.9±0.8 µm (N=50, mean value ± standard deviation). Colonies on MEA were 5.4 to 5.8 cm in diameter after growth for seven days at 20 to 25°C with a photoperiod of 12 h and 3.3 to 3.7 cm in diameter after growth for seven days in the dark at 22°C. These morphological observations led to the conclusion that the isolate may belong to the genus Phoma. To test this hypothesis, we performed a drop test with 5 M NaOH which is used routinely to check for the presence of a genus-specific metabolite. We observed a change in color, indicating a positive test result. The color change was even more pronounced on the plates incubated in the light, further confirming the presence of "metabolite E" (Boerema et al. 2004; Kövics et al. 2014). Next, DNA was extracted and PCR was performed with primers specific for ITS regions (GenBank MT397284), LSU (MT397285), rbb2 (MT414713) or tub2 (MT414712). Sequencing results of PCR products were used to create a combined phylogenetic tree, including sequences published previously (Chen et al. 2015). Our sequencing results together with the morphological observations clearly identified the fungal isolate to be Boeremia exigua var. exigua. The isolate is publicly available in the CBS collection of the Westerdijk Fungal Biodiversity Institute with the accession no. CBS 146730. Koch's postulates were fulfilled by inoculating a spore suspension of the isolate IPP1903 (5x105 ml-1 in 0.05% Tween 20 solution in distilled water) onto healthy primary leaves of twenty 14 days old soybean plants of the cultivar Abelina. While the mock-inoculated plants (inoculated with 0.05% Tween 20 solution in distilled water) stayed healthy, the inoculated plants developed lesions on the leaves after seven days. Six weeks after inoculation the fungus could be reisolated from cuttings of the infected leaves after surface-sterilization. Fungal colonies were confirmed to be B. exigua var. exigua by morphological examination and via NaOH drop test. To our knowledge, this is the first report of B. exigua var. exigua causing disease on commercially grown soybean in Germany.

On the current status of Phakopsora pachyrhizi genome sequencing.

Recent advances in the field of sequencing technologies and bioinformatics allow a more rapid access to genomes of non-model organisms at sinking costs. Accordingly, draft genomes of several economically important cereal rust fungi have been released in the last 3 years. Aside from the very recent flax rust and poplar rust draft assemblies there are no genomic data available for other dicot-infecting rust fungi. In this article we outline rust fungus sequencing efforts and comment on the current status of Phakopsora pachyrhizi (Asian soybean rust) genome sequencing.

In vivo assessment by Mach-Zehnder double-beam interferometry of the invasive force exerted by the Asian soybean rust fungus (Phakopsora pachyrhizi).

Asian soybean rust (Phakopsora pachyrhizi) causes a devastating disease in soybean (Glycine max). We tested the hypothesis that the fungus generates high turgor pressure in its hyaline appressoria to mechanically pierce epidermal cells. Turgor pressure was determined by a microscopic technique, called transmitted light double-beam interference Mach-Zehnder microscopy (MZM), which was developed in the 1960s as a forefront of live cell imaging. We revitalized some original microscopes and equipped them for modern image capturing. MZM data were corroborated by cytorrhysis experiments. Incipient cytorrhysis determined the turgor pressure in appressoria of P. pachyrhizi to be equivalent to 5.13 MPa. MZM data revealed that osmotically active sugar alcohols only accounted for 75% of this value. Despite having a lower turgor pressure, hyaline rust appressoria were able to penetrate non-biodegradable polytetrafluoroethylene (PTFE) membranes more efficiently than do melanized appressoria of the anthracnose fungus Colletotrichum graminicola or the rice blast fungus Magnaporthe oryzae. Our findings challenge the hypotheses that force-based penetration is a specific hallmark of fungi differentiating melanized appressoria and that this turgor-driven process is solely caused by metabolic degradation products. The appressorial turgor pressure may explain the capability of P. pachyrhizi to forcefully invade a wide range of different plants and may pave the way to novel plant protection approaches.

Phakopsora pachyrhizi, the causal agent of Asian soybean rust.

UNLABELLED: The plant pathogenic basidiomycete fungi Phakopsora pachyrhizi and Phakopsora meibomiae cause rust disease in soybean plants. Phakopsora pachyrhizi originated in Asia-Australia, whereas the less aggressive P. meibomiae originated in Latin America. In the New World, P. pachyrhizi was first reported in the 1990s to have spread to Hawaii and, since 2001, it has been found in South America. In 2004, the pathogen entered continental USA. This review provides detailed information on the taxonomy and molecular biology of the pathogen, and summarizes strategies to combat the threat of this devastating disease. TAXONOMY: Phakopsora pachyrhizi Syd. & P. Syd; uredial anamorph: Malupa sojae (syn. Uredo sojae); Domain Eukaryota; Kingdom Fungi; Phylum Basidiomycota; Order Uredinales; Class Urediniomycetes; Family Phakopsoraceae; Genus Phakopsora (http://www.indexfungorum.org). The nomenclature of rust spores and spore-producing structures used within this review follows Agrios GN (2005) Plant Pathology, 5th edn. London: Elsevier/Academic Press. HOST RANGE: In the field, P. pachyrhizi infects leaf tissue from a broad range (at least 31 species in 17 genera) of leguminous plants. Infection of an additional 60 species in other genera has been achieved under laboratory conditions. DISEASE SYMPTOMS: At the beginning of the disease, small, tan-coloured lesions, restricted by leaf veins, can be observed on infected soybean leaves. Lesions enlarge and, 5-8 days after initial infection, rust pustules (uredia, syn. uredinia) become visible. Uredia develop more frequently in lesions on the lower surface of the leaf than on the upper surface. The uredia open with a round ostiole through which uredospores are released.

Transgenic suppression of cell death limits penetration success of the soybean rust fungus Phakopsora pachyrhizi into epidermal cells of barley.

The basidiomycete Phakopsora pachyrhizi (P. pachyrhizi) causes Asian soybean rust, one of the most devastating plant diseases on soybean. When inoculated on the nonhost barley P. pachyrhizi caused only very small necrotic spots, typical for an incompatible interaction, which involves a hypersensitive cell death reaction. A microscopic inspection of the interaction of barley with P. pachyrhizi revealed that the fungus germinated on barley and formed functional appressoria on epidermal cells. The fungus attempted to directly penetrate through periclinal cell walls but often failed, arrested in plant cell wall appositions that stained positively for callose. Penetration resistance depends on intact ROR1(REQUIRED FOR mlo-SPECIFIED RESISTANCE 1) and ROR2 genes of barley. If the fungus succeeded in penetration, epidermal cell death took place. Dead epidermal cells did not generally restrict fungal development but allowed for mesophyll invasion, which was followed by mesophyll cell death and fungal arrest. Transient or stable over expression of the barley cell death suppressor BAX inhibitor-1 reduced both epidermal cell death and fungal penetration success. Data suggest that P. pachyrhizi provokes a programmed cell death facilitating fungal entry into epidermal cells of barley.

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.