First report of Neophysopella vitis causing leaf rust on Parthenocissus semicordata in Guangdong Province, China.

In April 2022, leaf rust disease of Parthenocissus semicordata was discovered in the urban greenbelt in Guangzhou city, China (23.06°N, 113.16°E). The disease incidence was 70% and disease severity was 75%. Chlorotic spots and red-brown necrotic flecks were present on the upper surface of infected leaves, and orange uredinia were distributed on the lower surface (Fig. 1 a-c). Two representative disease plants were collected as voucher specimens and dried, then deposited in Mycological Herbarium of the Zhongkai University of Agriculture and Engineering (MHZU GR0413, MHZU GR0414). Microscopic examination of the pustules of the samples revealed the presence of uredial paraphyses and urediniospores (Fig.1 d-f). Uredial paraphyses were hyaline, incurved, length 20-25 µm and dorsal wall 4.5-8.5 µm thick. Urediniospores were subglobose to ovoid, 16-24.5 × 10.5-17 µm. The wall of the urediniospore was hyaline or pale yellow, echinulate, and 1.0-2.0 µm thick. Telial structures were not observed. The morphological characteristics of uredial paraphyses and urediniospores were consistent with Yoshitaka's description of Neophysopella vitis (Yoshitaka 2000). Ten uredinia were picked using a sterile tweezer and the FlaPure Plant DNA Extraction Kit (Genesand Biotech Co., Ltd) was used to extract genomic DNA. Primers ITS4-rust (5'-CAGATTACAAATTTGGGCT-3') /ITS 5u (5'-CAAGGTTTCTGTAGGTG-3') were used to amplify internal transcribed spacer (ITS) and Rust2inv (5'-GATGAAGAACACAGTGAAA-3') /LR6 (5'-CGCCAGTTCTGCTTACC-3') to amplify large subunit (LSU) rDNA regions (Zhao et al. 2021). The sequences were submitted to NCBI (ITS: OQ991182 OQ991183, LSU: OQ979612 OQ979613), and blastn analyses of ITS and LSU showed 99.81% and 99.84% identity with the sequences from N. vitis (ITS: OQ304336, LSU: OM420266), respectively, found in GenBank. ITS combined with LSU sequences from the current study and reference sequences from Zhao et al. (2021) were used to construct Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees using CIPRES website. ML and BI phylogenetic trees showed that the sequences from the current study grouped with N. vitis, with bootstrap support and posterior probability of 98% and 1.0, respectively (Fig. 2). Morphological and molecular analyses confirmed that the rust fungus was N. vitis. In the pathogenicity test, urediniospores were picked from fresh diseased leaves with sterile tweezers, prepared into spore suspensions (1.0 × 106 urediniospores/ml), and sprayed on a one-week-old leaf from three healthy of potted plants of P. semicordata, while three other healthy leaves were sprayed with sterile water as control. Each treated leaf was wrapped in a plastic bag and incubated in the dark at 25℃ for 48 h after which plastic bags were removed and the plant were moved to a greenhouse at 25℃. At 15 days after inoculation, symptoms and the morphology of urediniospores were confirmed to be the same as those observed in the field collection and no symptoms or uredinia were observed in the control group (Fig.1 g, h). Yoshitaka first discovered leaf rust on P. semicordata caused by N. vitis in Nepal(Yoshitaka 2000). To our knowledge, this is the first report of N. vitis causing leaf rust on P. semicordata in China. P. semicordata is often used as an ornamental plant for exterior wall decoration in urban landscaping and court. The occurrence of this disease can lead to the decline of the P. semicordata leaves and the impairment of the plants aesthetically.

Dynamics of Puccinia striiformis f. sp. tritici Urediniospores in Longnan, a Critical Oversummering Region of China.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Erikss. (Pst), is a devastating disease resulting in yield reduction. Because the temperature limits the overwintering and oversummering of Pst, it cannot complete the whole year cycle in most areas of China. Longnan, located in the southeast of Gansu Province, is one of the annual cycle areas of Pst, which can supply urediniospores in autumn to eastern wheat-growing areas in China. In this study, a TaqMan real-time quantitative PCR (TaqMan-qPCR) detection system for Pst urediniospores was established, and the detection limit was a single urediniospore. The dynamics of Pst urediniospores in Longnan were monitored by spore trapping and TaqMan-qPCR for 3 years. Meanwhile, the meteorological conditions including air temperature, relative humidity, and precipitation were recorded. Results showed that Pst urediniospores can be captured from March to December, and two peaks of urediniospore density appeared in May and June, respectively. The density of urediniospore is closely related to temperature and precipitation from March to June. In addition, we found that the density of Pst urediniospore had the peak value when the average air temperature was 10 to 21°C, and the relative humidity was 60 to 85% from May to June. The exponential model could describe the variation of Pst urediniospore density based on average temperature and precipitation from March to June. It is worth mentioning that the exponential model based on average temperature 7 days before spore capture has a great advantage in predicting the urediniospore density in the air. This study laid a foundation for establishment of a prediction model for wheat stripe rust based on the density of urediniospores and meteorological factors.

Electron microscopy of the uredinial and telial stages of the rust fungus, Puccinia imperatae.

The purpose of this work was to assess the morphology and ontogeny of Puccinia imperatae urediniospores and teliospores growing on its host, Imperata cylindrica, for the first time using scanning (SEM) and transmission (TEM) electron microscopy. The first evidence of uredinial development is the aggregation of hyphae in host intercellular spaces under the epidermis to form an uredinial initial. Uredinial primordia become evident as compact masses of fungal hyphae. The mycelia are dikaryotic and originated from dikaryotic urediniospores, which infect a new host. Dikaryotic sporogenous cells begin to form from dikaryotic mycelium as the uredinium develops further. The fundamental cell in the urediniospore and teliospore formation is the sporogenous cell. The teliospores with their persistent pedicels arise from dense dikaryotic sporogenous tissue, which later becomes highly vacuolated. The sporogenous cell forms a spore bud, which later divides into a pedicel and teliospore mother cell with two nuclei that produce the mature teliospore. SEM examination revealed that the urediniospores are echinulate bearing conical spines on their surfaces, sometimes curving near their tips, and the echinulate ornaments are distributed over the entire urediniospore surface. SEM examination also indicated that the teliospore surface shows rugose ornamentations. This investigation proved that the morphology of the uredinia of different Puccinia species is not related to the host species, but to the rust species. The possibility of using this fungus as a biological control agent for the noxious weed, Imperata cylindrica, was also discussed.

First report of stem rust, caused by Puccinia graminis, and crown rust, caused by Puccinia coronata var. avenae f. sp. avenae, on tall fescue (Festuca arundinacea) in Georgia, USA.

Stem rust, caused by Puccinia graminis, and crown rust, caused by P. coronata, are common rust diseases on cool-season grasses (Karakkat et al. 2018), for which long-distance spore dispersal was recorded in northern US (Harder and Haber 1992). During the summers of 2019 and 2020, severe infection of stem rust and crown rust was observed on > 60% of tall fescue (Festuca arundinacea) germplasm plants in a breeding nursery located at the University of Georgia, Griffin GA. Rust-infected leaves first presented uredinia pustules, then black telia towards the end of the season. The uredinia pustules of stem rust and crown rust were brick-red and, yellow and arranged along the host veins, respectively. The urediniospores were one-celled, round to ovoid and measured from 20.75±2.44 µm (crown rust) to 27±3.60 µm long (stem rust). The teliospores were two-celled and measured from 45.75±10.14 µm (stem rust) to 51.60±4.0 µm long (crown rust) (Leonard et al. 2005; Cummins 1971). Urediniospores of both rusts were collected from infected plants in the field in April of 2020 using a Piston vacuum pump (Welch by Gardner Denver Ltd.) and stored at -80 °C in 1.5 ml Eppendorf tubes. Genomic DNA was extracted by grinding the urediniospores in liquid nitrogen using mortar and pestle, followed by the cetyltrimethylammonium bromide method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using the ITS5-ITS4 primers (White et al. 1990). BLASTn and phylogenetic analyses revealed that the sequence of stem rust (GenBank acc. no. MW430963) and crown rust (GenBank acc. no. MW431324) pathogens had >99% similarity with P. graminis (GenBank acc. no. HQ317538) and P. coronata var. avenae f. sp. avenae (clade V; Liu and Hambleton 2013) (GenBank acc. no. EU014044), respectively. Pathogenicity tests were conducted on the tall fescue cultivar 'Bandit'. For each rust, 12 pots (10 cm × 10 cm) were planted, each containing 13 seeds in a Sungro professional growing mix soil (Sun Gro Horticulture Distribution Inc.). The plant materials were kept in the greenhouse at 20°C/ 25°C (night/day),15-hrs of light, and watered twice a week for 4-weeks. Urediniospores were recovered from -80°C and allowed to acclimate at room temperature for 1 h. For each rust, 20 ml of suspension containing 1×105 urediniospores ml-1 and 5 µl of Tween-twenty (Agdia Inc. Elkhart, IN) were used to inoculate 6 pots; while 6 control pots were sprayed with sterile water. After inoculation, plants were allowed to dry for 1 h and then transferred to a dark chamber at 20°C and 90% of humidity for 12-15 h. At 10-days post inoculation, all inoculated plants developed rust symptoms identical to those observed in the field, whereas control plants had no symptoms. Stem and crown rust pathogens were re-isolated from the artificially inoculated tall fescue plants. Based on form, size, color and numbers of cells forming the spores, a 1947 Festuca elatior specimen from Georgia mentioning Puccinia coronata (Hanlin 1966), held at the Julian H. Miller Mycological Herbarium (Catalog No. GAM00013162), was discarded as an earlier record of P. coronata var. avenae and could have been misdiagnosed. Due to the fragile integrity of the original infected plant sample as well as the incipient infection, DNA identification was unsuccessful. To our knowledge, this is the first morphological, genetic and taxonomic report of P. graminis and P. coronata var. avenae f. sp. avenae on tall fescue in Georgia, USA.

Alteraciones histopatológicas causadas por la roya Puccinia nakanishikii (Pucciniales: Pucciniaceae) en plantas de Cymbopogon citratus (Poaceae) / Histopathological alterations caused by Puccinia nakanishikii rust (Pucciniales: Pucciniaceae) in Cymbopogon citratus (Poaceae) plants

Introducción: Los aspectos histopatológicos e histoquímicos relacionados con el ataque de royas en plantas, así como su relación con los diferentes estados espóricos, son escasos en la literatura científica. Objetivos: Describir y analizar los aspectos histopatológicos e histoquímicos en Cymbopogon citratus y su relación con los diferentes estados espóricos de la roya Puccinia nakanishikii. Métodos: Durante abril y agosto 2013 se recolectaron hojas sanas e infectadas con Puccinia nakanishikii en la escarpa noroccidental de la meseta de Bucaramanga-Colombia. Las muestras con lesiones en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en parafina (5-7 µm) fueron teñidas con Safranina-azul de Alcian y azul de Alcian-Hematoxilina. En tanto que las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. También se elaboraron secciones a mano alzada para análisis de autofluorescencia. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), las muestras se fijaron en Glutaraldehído, se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Las hojas son por lo general hipostomáticas, con células epidérmicas largas y cortas formando filas paralelas y con la presencia de tricomas unicelulares espinosos y microtricomas. La superficie abaxial está cubierta por una densa capa de ceras epicuticulares y la adaxial está formada por agrupaciones de células buliformes y células epidérmicas de contorno rectangular o cuadrado. En el mesófilo no hay diferenciación entre parénquima de empalizada y esponjoso y su anatomía refleja el metabolismo C4 presente en esta gramínea. Se observó la formación de urediosoros y teliosoros hipófilos. Las urediosporas son la fase de reinfección y estas tienen de 4-5 poros germinativos ecuatoriales y su pared es equinulada. Las teliosporas son de pared lisa y de pedicelo persistente. Las urediosporas forman tubos de geminación por lo general sobre la superficie abaxial de la hoja y se desarrollan en dirección de los estomas, por donde penetran al interior del mesófilo. No se observó la presencia de apresorios. La epidermis se desprende y levanta por del desarrollo de las urediosporas y las paráfisis capitadas, a medida que el urediosoro crece. Con el avance de la infección, los tejidos fotosintéticos se desorganizan, pierden la autofluorescencia de la clorofila y las células sufren necrosis. Posteriormente, los tejidos vasculares se fragmentan y colapsan. Para este momento, la infección se ha extendido sobre toda la lámina foliar llevando a la muerte de la hoja y defoliación de la planta. Durante etapas avanzadas de la infección en los urediosoros se observaron picnidios, probablemente del hiperparásito Sphaerellopsis, asociados estrechamente a los tejidos infectados por la roya. Conclusiones: Puccinia nakanishikii se desarrolla sobre las hojas de Cymbopogon citratus produciendo urediosoros y teliosoros. Las urediosporas son la fase de reinfección, y las teliosporas solo se observaron en etapas avanzadas de la infección. La epidermis y los tejidos fotosintéticos son severamente afectados por la necrosis celular. En etapas avanzadas de la infección los tejidos vasculares se ven afectados.

Introduction: Histopathological and histochemical aspects linked to the attack of fungal rusts to plants, as well as its relation with the different spore stages are topics rather scarce in the scientific literature. Objective: To describe and analyze the histopathological and histochemical aspects of Cymbopogon citratus and its relation with the different stages of the spores from the rust fungi Puccinia nakanishikii. Methods: During the months April and August 2013, leaves healthy and infected by Puccinia nakanishikii were collected in the Northwestern scarp of the Bucaramanga-Colombia plateau. The samples with injuries on diverse developmental stages were fixated and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained from paraffin (5-7 µm) were stained with Safranin-Alcian blue and Alcian blue-Hematoxylin. On the other hand, sections obtained from resin (0.5 µm) were stained with Toluidine blue. Further, freehand sections were obtained for an autofluorescence analysis. The observations and photographic record were done via photonic microscope and epifluorescence microscope. For the observations via scanning electron microscopy (SEM), the samples were fixated in Glutaraldehyde, dehydrated with 2,2 dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The leaves are generally hypostomatic, with long and short epidermic cell forming parallel rows and showing unicellular prickle trichomes and micro-trichomes. The abaxial surface is covered by epicuticular wax forming a dense layer. The adaxial epidermis is formed by groupings of bulliform cells and epidermal cells with rectangular or squared contour. In the mesophyll, there is no differentiation between palisade and spongy parenchyma, its anatomy reflects the C4 metabolism. The formation of uredosori and teliosori both hypophyllous was observed. Urediniospores are the reinfecting agents phase, they have 4-5 equatorial germ pores and echinulate wall. Teliospores have smooth wall and a persistent pedicel. The urediniospores form a germ tube, generally on the abaxial leaf surface, these tubes develop towards the stomata reaching the mesophyll interior. No appressorium were observed. The epidermis limiting the uredosorus detaches due the development and pressure that exert both the urediniospores and capitate paraphyses. As the infection progresses, autofluorescense of the chlorophyll is lost and the cells undergo necrotic processes. Afterwards, the phloem collapses and the xylem becomes slightly disorganized. At this moment, the infection is extended along the whole leaf blade, resulting in the leaf death and the plant defoliation. On advanced stages of the infection, the uredosori showed pycnidia, probably belonging to the hyperparasite Sphaerellopsis, these structures were closely associated to the rust infected tissues. Conclusions: Puccinia nakanishikii develops on the leaves of Cymbopogon citratus producing uredosori and teliosori. Urediniospores are the reinfective stage, teliospores were only observed at late stages of the infection. The epidermis and photosynthetic tissue are severely affected by cell necrosis. The vascular tissues are deeply affected on the advances stages of the infection.

Additive Manufacturing of Devices Used for Collection and Application of Cereal Rust Urediniospores.

Optimized inoculation procedures are an important consideration in achieving repeatable plant infection when working with biotrophic rust fungi. Several plant pathology laboratories specializing in rust research employ a system where the collection and application of fungal spores are accomplished using an exchangeable gelatin capsule. Urediniospores are collected from erumpent pustules on plant surfaces into a capsule fitted to a cyclone collector controlled by a vacuum pump. By adding light mineral oil to the same capsule, the spore suspension is then sprayed onto plants by means of a dedicated atomizer (inoculator) connected to an air pressure source. Although devices are not commercially available, modern day technologies provide an opportunity to efficiently design and manufacture collectors and inoculators. Using a process called Additive Manufacturing (AM), also known as "3D printing," the bodies of a collector and inoculator were digitally designed and then laser-sintered in nylon. Depending on availability, copper or aluminum tubes were fitted to the bodies of both devices afterward to either facilitate directed collection of spores from rust pustules on plant surfaces or act as a siphon tube to deliver the spore suspension contained in the capsule. No statistical differences were found between AM and metal inoculators for spray delivery time or spore deposition per unit area. In replicated collection and inoculation tests of wheat seedlings with urediniospore bulks or single pustule collections of Puccinia triticina and P. graminis f. sp. tritici, the causal organisms of leaf rust and stem rust, consistent and satisfactory infection levels were achieved. Immersing used devices in acetone for 60 s followed by a 2 h heat treatment at 75°C produced no contaminant infection in follow-up tests.

Development of a rapid RNA extraction procedure from urediniospores of the leaf rust fungus, Puccinia triticina.

Obtaining high quality RNA in good quantities is often a requirement for plant-pathogen interaction studies, so it becomes very essential that a highly efficient method should be deployed to isolate RNA from minute quantities of fungal spores. The methods available to date, either require a high quantity of spores or the use of expensive chemicals. The protocol discussed here for RNA isolation from Puccinia triticina pathotype 77-5 urediniospores utilizes TRI Reagent as extraction buffer that is widely used for RNA isolation from plant tissues. Urediniospores have a tough cell wall as compared to other plant cells. Therefore, the protocol was optimized keeping the primary focus on quickly disrupting cell walls. Two different methods, one using a combination of liquid nitrogen and ultrasonic water-bath and the other method using micro-homogenizer were utilized for crushing the spores in the present study. The developed methods do not utilize mortar and pestle, instead they promote direct crushing of urediniospores in tubes; thereby minimizing sample loss and enhancing quality.

Saprophytic growth of the alder rust fungus Melampsoridium hiratsukanum on artificial media.

The first axenic culture of a free living saprophytic stage of the exotic rust fungus Melampsoridium hiratsukanum is reported. Colonies were obtained from one-celled, dikaryotic urediniospores on eight nutrient media out of twelve. Modified Harvey and Grasham (HG) and Schenk and Hildebrandt (HS) media HG1 and SH1 and their bovine serum albumin (BSA)-enriched derivatives gave abundant mycelial growth, but modified Murashige and Skoog (MS) QMS media and their BSA-enriched modifications performed poorly, colony growth being low on QMS-1 and QMS1+BSA, and nil on QMS-5 and QMS-6, with or without BSA. Colonies initially grew poorly when subcultured for one month in purity, but much better after re-transfer to fresh media later: presumably because only the most exploitative genotypes survived, best able to cope with an uncongenial medium. Stabilised cultures survived, and remained vegetative, but only few reproductive colonies produced spore-like bodies. Though the agarised medium remains an inhospitable environment for this biotrophic parasite, it is shown that non-living media can nevertheless sustain the growth and sporulation of this fungus outside its natural hosts and habitat. Axenic culture promises important advances in basic and applied research on this rust, leading to a better understanding of its nutrition, metabolism, diversity and pathogenicity.

Anatomia foliar de soja infectada por Phakopsora pachyrhizi H. Sydow & Sydow e tratadas com extratos vegetais / Leaf anatomy of soybean infected with Phakopsora pachyrhizi H. Sydow & Sydow and treated with plant extracts

Folhas de Glycine max (L.) Merril,infectadas pelo fungo Phakopsora pachyrhizi H. Sydow & Sydow e tratadas com extratos vegetais foram avaliadas, visando determinar in vivo as modificações anatômicas nas diferentes estruturas/tecidos foliares, além de reconhecer prováveis mecanismos de defesa. Folhas de soja cultivar 181 provenientes do quinto nó foram inoculadas com fungo e tratadas com diferentes extratos vegetais, água e álcool 70%. Para comparação foram analisadas a anatomia das folhas sadia e infectada e realizadas medidas nas estruturas/tecidos foliares. Na folha infectada, observou-se destruição da epiderme e parênquima lacunoso, visível proliferação de tricomas e cutícula espessada, principalmente na face abaxial. Observou-se a presença de compostos fenólicos nas células da epiderme quando rompida, em função do crescimento micelial. Nas folhas infectadas e tratadas com os extratos vegetais de Azadirachta indica, Maytenus ilicifolia e Allium sativum, as estruturas/tecidos vegetais apresentaram aumento de espessura por alongamento celular.

Glycine max (L.) Merril leaves, infected by the fungus Phakopsora pachyrhizi H. Sydow & Sydow and treated with plant extracts, were evaluated with the aim of determining in vivo the anatomical modifications in different leaf structures and of identifying probable defense mechanisms. Leaves from the 181 soybean cultivar originated from the fifth node were inoculated with the fungus and treated with different plant extracts, water and alcohol at 70%. For comparison, the anatomy of the healthy and infected leaves was analyzed and the leaf structures were measured. In the infected leaf, there was destruction of the epidermis and lacunar parenchyma, apparent trichome proliferation and denser cuticle, especially on the abaxial surface. There were also phenolic compounds in ruptured epidermis cells, due to mycelium growth. In the infected leaves treated with Azadirachta indica, Maytenus ilicifolia and Allium sativum extracts, the plant tissues had an increase in thickness due to cell elongation.

Métodos de inoculação de Phakopsora euvitis Ono em Vitis labrusca L / Inoculation methods of Phakopsora euvitis Ono in Vitis labrusca L

A ferrugem é uma das principais doenças da videira, com ocorrência registrada em diversas regiões produtoras. Neste trabalho, foram avaliados três métodos de inoculação de Phakopsora euvitis em mudas de videira 'Isabel' com o objetivo de selecionar um método para determinação da patogenicidade. Mudas oriundas de bacelos de videira foram inoculadas com esporos de P. euvitis e mantidas sob temperatura entre 25±4°C e UR 85±10 por cento durante 13 dias. O delineamento experimental foi inteiramente casualizado, com 10 repetições. Os tratamentos foram compostos por: T1 - pincelamento de esporos; T2 - pulverização com suspensão de esporos com 2,7x10³ esporos mL-1; T3 - discos de folhas de videira com sintomas afixados em cinco folhas por planta; e T4 - Testemunha. O método do pincelamento de esporos foi o mais eficiente, com início dos sintomas a partir do quinto dia e, após 13 dias, 100 por cento das folhas apresentavam sinais do patógeno.

Rust is one of the most important diseases in grapevine, with occurrence recorded in many production areas. In this research it was evaluated three methods of Phakopsora euvitis inoculation on grapevine seedlings cv. 'Isabel' with the objective of selecting an inoculation method for rapid pathogenicity determination. Grapevine seedlings were inoculated with P. euvitis and maintained under conditions of 25±4C and UR 85±10 percent for 13 days. The experimental design was completely randomized, with 10 replications. The treatments were: T1 - brush of pathogen spores; T2 - spores suspension spraying (2.7x10³ spores mL-1); T3 - disks of grapevine leaves with symptoms stuck in five leaves per plant and T4 - control. The spore brushing method was the most efficient, showing early symptoms at five days, and 100 percent of the leaves showing pathogen symptoms 13 days after treatment.