Sequencing Herbarium Specimens of a Common Detrimental Plant Disease (Powdery Mildew)

Powdery mildew (Erysiphaceae) is a detrimental plant disease that occurs on a variety of economically important crops. Powdery mildew consists of over 873 species of fungal pathogens that affect over 10,000 plant species. Genetic identification of powdery mildew is accomplished using the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal RNA gene cluster. The ITS and LSU regions of powdery mildews can be useful in ecological, epidemiological, phylogenetic, and taxonomic investigations. However, sequencing these regions is not without its challenges. For example, powdery mildew sequences are often contaminated with plant and/or fungal DNA. Also, there tends to be a limited amount and older specimens’ DNA can fragment over time. The success of sequencing powdery mildew often depends on the primers used for running polymerase chain reaction (PCR). The primers need to be broad enough that they match the majority of powdery mildew DNA yet specific enough that they do not align with other organisms. A review of the taxonomy and phylogeny of the powdery mildews is presented with an emphasis on sequencing the ITS + LSU genomic regions. Additionally, we introduce a new nested primer protocol for sequencing powdery mildew herbarium samples that includes six new powdery mildew-specific primers. The new sequencing protocol presented allows specimens up to 130 years old to be sequenced consistently. Sequencing herbarium specimens can be extremely useful for addressing many ecological, epidemiological, phylogenetic, and taxonomic problems in multiple plant pathogenic systems including the powdery mildews.

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Using PCR to Distinguish Diaporthe phaseolorum and Phomopsis longicolla from Other Soybean Fungal Pathogens and to Detect Them in Soybean Tissues

Plant Disease ◽

10.1094/pdis.1997.81.10.1143 ◽

1997 ◽

Vol 81 (10) ◽

pp. 1143-1149 ◽

Cited By ~ 44

Author(s):

A. W. Zhang ◽

G. L. Hartman ◽

L. Riccioni ◽

W. D. Chen ◽

R. Z. Ma ◽

...

Keyword(s):

Fungal Pathogens ◽

Nuclear Ribosomal Dna ◽

Specific Primers ◽

Phomopsis Longicolla ◽

Tissue Samples ◽

Diaporthe Phaseolorum ◽

Pcr Products ◽

Fungal Dna ◽

Dna Fragment ◽

Polymerase Chain

Restriction fragment length polymorphism analyses of polymerase chain reaction (PCR) amplified DNA were used to distinguish Diaporthe phaseolorum and Phomopsis longicolla isolates from other soybean fungal pathogens. Primers made to the conserved sequences of nuclear ribosomal DNA amplified the internal transcribed spacer (ITS) regions of D. phaseolorum var. meridionalis and P. longicolla. The PCR products were cloned and then sequenced. Specific-primers, Phom.I and Phom.II, were designed from the polymorphic regions of D. phaseolorum and P. longicolla isolates from soybean to distinguish them from other soybean fungal pathogens. These ITS-derived primers amplified a 337-bp-specific DNA fragment from P. longicolla, D. phaseolorum var. meridionalis, D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and Phomopsis spp. from 20 different hosts. No amplified product was observed using DNA of seven other soybean fungal pathogens or soybean DNA. The detection limit of PCR using primers Phom.I and Phom.II was 2.5 × 10-7 dilution of fungal DNA extracted from samples of 10 pooled seeds and as low as a 1:15 (Phomopsis:soybean) ratio when using 10 ng of DNA per μl from each P. longicolla and soybean. PCR did not produce products using primers Phom.I and Phom.II with DNA extracted from noninfected seeds, but specific bands were observed from samples of 10 pooled seeds and from individually infected seeds. A specific band was observed as well from DNA extracts of tissue samples from symptomless plants inoculated with P. longicolla and D. phaseolorum var. sojae.

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Outbreak of Powdery Mildew Caused by Erysiphe cruciferarum on Spider Flower (Cleome hassleriana) in Italy

Plant Disease ◽

10.1094/pdis-93-9-0963c ◽

2009 ◽

Vol 93 (9) ◽

pp. 963-963 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

M. L. Gullino

Keyword(s):

Powdery Mildew ◽

Its Region ◽

Herbarium Specimens ◽

Pathogenicity Test ◽

Public Park ◽

Potted Plants ◽

Powdery Mildews ◽

Erysiphe Cruciferarum ◽

Average Size ◽

Germ Tubes

Spider flower (Cleome hassleriana L. [synonym = C. spinosa]) is used as a tall border in parks and gardens worldwide. During July 2008, severe outbreaks of a powdery mildew were observed in a public park in Torino (northern Italy). Leaves were covered with dense, white hyphae and conidia, especially on the adaxial surface. Hyphae were also present on petioles and fruits, but not on petals and stems. As the disease progressed, infected leaves turned chlorotic, curled, and wilted. Conidia were hyaline, cylindrical, single, and measured 31.1 to 48.2 × 12.9 to 17.6 μm (average 37.1 × 15.6 μm). Germ tubes terminating in a moderately lobed appressorium were produced terminally. The cylindrical foot cells of the erect condiophores were 19.2 to 27.8 × 6.5 to 8.6 μm (average 23.3 × 7.7 μm). Fibrosin bodies were absent. Chasmothecia were observed mostly on the lower surfaces of leaves. At maturity, they were dark amber and spherical with a diameter of 92.9 to 151.0 μm (average 121.4 μm). Each chasmothecium contained six stalked asci (average size 63.7 × 35.9 μm). Each ascus contained four ellipsoid ascospores that measured 17.3 to 26.4 × 10.9 to 15.6 μm (average 23.3 × 12.8 μm). The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced (1). The 602-bp sequence was deposited in GenBank under the Accession No. GQ149478 and was 99% similar to that of Erysiphe cruciferarum (Accession No. EU140958). As proof of pathogenicity, diseased leaves of C. hassleriana were pressed against leaves of three healthy 4-month-old potted plants of the same species for 10 min. Three noninoculated plants served as controls. Inoculated and noninoculated plants were maintained in a greenhouse at 22 to 25°C in isolation. After 11 days, typical powdery mildew colonies developed on inoculated plants. Noninoculated plants did not develop symptoms. The pathogenicity test was repeated once. Powdery mildew on C. hassleriana caused by E. cruciferarum was reported in Italy (2) but the pathogen was not characterized. Herbarium specimens are deposited at AGROINNOVA Collection, University of Torino, Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena, Germany, 1995.

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‘Phytopathological strolls’ in the dual context of COVID-19 lockdown and IYPH2020: transforming constraints into an opportunity for public education about plant pathogens

10.1101/2021.02.03.429201 ◽

2021 ◽

Author(s):

Frédéric Suffert ◽

Muriel Suffert

Keyword(s):

Fungal Pathogens ◽

Plant Disease ◽

Plant Pathogens ◽

Digital Tools ◽

Pathogen Identification ◽

Open Spaces ◽

Powdery Mildews ◽

Fungal Plant Pathogens ◽

Large Audience ◽

The Times

AbstractThe experience presented here relates to 2020, a particularly timely year for plant disease-related communication (‘International Year of Plant Health’ IYPH2020), but also a unique year because of the COVID-19 pandemic. Our goal was to illustrate the diversity and beauty of fungal plant pathogens through a naturalist approach that could be followed by any amateur. We achieved this end through ‘phytopathological strolls’, in which we observed and determined the origin of symptoms on diseased plants found in our garden, in the local streets, in nearby open spaces, and sharing this matter with a broad public. The lockdown imposed in France created an additional motivation to take up the challenge, and to involve our children, even under strong constraints, such as movement restrictions. We observed and described fungal pathogens through hundreds of photographs, shared our findings with a large audience on Twitter, and received feedback. The material used was deliberately simple and transportable: a digital reflex camera, an old microscope, a mobile phone, some books and an Internet connexion. Between March 17 March and December 15, 2020 we found 148 plant pathogens, including 72 rusts, 22 powdery mildews and 22 septoria-like diseases. We discuss here the importance of promoting searches for plant pathogens, their description and conservation, through a combination of classical approaches and digital tools in tune with the times, such as Twitter, by treating pathogen identification like a detective game and, more surprisingly, by making use of the addictive nature of collection approaches, drawing a parallel with Pokémon Go.

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Fungicide Resistance in Powdery Mildew Fungi

Microorganisms ◽

10.3390/microorganisms8091431 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1431

Author(s):

Alejandra Vielba-Fernández ◽

Álvaro Polonio ◽

Laura Ruiz-Jiménez ◽

Antonio de Vicente ◽

Alejandro Pérez-García ◽

...

Keyword(s):

Powdery Mildew ◽

Fungal Pathogens ◽

Molecular Mechanisms ◽

Resistance Development ◽

Development Of Resistance ◽

Powdery Mildews ◽

Powdery Mildew Fungi ◽

Sterol Demethylation Inhibitors ◽

Chemical Groups ◽

Multiple Chemical

Powdery mildew fungi (Erysiphales) are among the most common and important plant fungal pathogens. These fungi are obligate biotrophic parasites that attack nearly 10,000 species of angiosperms, including major crops, such as cereals and grapes. Although cultural and biological practices may reduce the risk of infection by powdery mildew, they do not provide sufficient protection. Therefore, in practice, chemical control, including the use of fungicides from multiple chemical groups, is the most effective tool for managing powdery mildew. Unfortunately, the risk of resistance development is high because typical spray programs include multiple applications per season. In addition, some of the most economically destructive species of powdery mildew fungi are considered to be high-risk pathogens and are able to develop resistance to several chemical classes within a few years. This situation has decreased the efficacy of the major fungicide classes, such as sterol demethylation inhibitors, quinone outside inhibitors and succinate dehydrogenase inhibitors, that are employed against powdery mildews. In this review, we present cases of reduction in sensitivity, development of resistance and failure of control by fungicides that have been or are being used to manage powdery mildew. In addition, the molecular mechanisms underlying resistance to fungicides are also outlined. Finally, a number of recommendations are provided to decrease the probability of resistance development when fungicides are employed.

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First Report of Powdery Mildew Caused by Golovinomyces orontii (Erysiphe orontii) on Lamium galeobdolon in Italy

Plant Disease ◽

10.1094/pdis-91-5-0635b ◽

2007 ◽

Vol 91 (5) ◽

pp. 635-635 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

D. Minerdi ◽

M. L. Gullino

Keyword(s):

Powdery Mildew ◽

Ground Cover ◽

Herbarium Specimens ◽

Pathogenicity Test ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Golovinomyces Orontii ◽

Powdery Mildews ◽

Blastn Analysis ◽

Ornamental Species

Lamium galeobdolon L. (Labiatae) is a common ornamental species that grows in shade areas and often used as a ground cover in gardens. During the summer of 2006, severe outbreaks of a previously unknown powdery mildew were observed on all Lamium spp. plants in some gardens near Biella (northern Italy). Both surfaces of the leaves of affected plants were covered with dense, white mycelia and conidia. As the disease progressed, infected leaves turned yellow and died. Mycelia and conidia also were observed on stems and flowers. Conidia were hyaline, ellipsoid, borne in short chains (with a maximum of five conidia per chain), and measured 29 to 37 × 16 to 20 μm (average 33 × 18 μm). Conidiophores, 91 to 104 μm (average 96 μm) long, showed the foot cell measuring 28 to 49 × 9 to 11 μm (average 38 × 10 μm), followed by three shorter cells measuring 14 to 26 × 9 to 15 μm (average 21 × 11 μm). Fibrosin bodies were absent. Chasmothecia were not observed in the collected samples. The internal transcribed spacer region (ITS) of rDNA was amplified using the primers ITS4/ITS6 (4) and sequenced. BLASTn analysis (1) of the 436 bp obtained showed an E-value of 0.0 with Golovinomyces orontii (Erysiphe orontii.) (3). The nucleotide sequence has been assigned GenBank Accession No. EF 121871. Inoculations were made by gently pressing diseased leaves onto leaves of five healthy L. galeobdolon plants. Five noninoculated plants served as controls. Inoculated and noninoculated plants were maintained in a greenhouse at temperatures between 15 and 28°C. After 10 days, typical powdery mildew colonies developed on inoculated plants. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of powdery mildew on L. galeobdolon caused by G. orontii in Italy. Blumer (2) was able to reproduce powdery mildew symptoms on L. galeobdolon using populations from cucumber, while Braun (3) reported L. galeobdolon as a possible host of E. orontii. Herbarium specimens of this disease are available at AGROINNOVA Collection, University of Torino, Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) S. Blumer. Ber. Schweiz. Bot. Ges. 62:384, 1952. (3) U. Braun. A Monograph of the Erysiphaceae (Powdery Mildews). Cramer, Berlin, GDR, 1987. (4) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997.

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Characteristics of powdery mildew and its importance for wheat grown in Poland

Plant Protection Science ◽

10.17221/99/2019-pps ◽

2020 ◽

Vol 56 (No. 3) ◽

pp. 141-153

Author(s):

Aleksandra Pietrusińska ◽

Anna Tratwal

Keyword(s):

Powdery Mildew ◽

Resistance Genes ◽

Host Species ◽

Chemical Control ◽

Plant Disease ◽

Common Procedure ◽

Breeding Programs ◽

Powdery Mildews ◽

Fungal Plant Disease ◽

High Degree

Powdery mildew of grasses and cereals (Blumeria graminis) is a fungal plant disease which is caused by species of fungi from the Erysiphaceae order. B. graminis is a biotrophic parasite, biologically diverse parasite with a high degree of specialization in certain host species and with numerous physiological breeds adapted to different varieties of a particular host species. In Poland, powdery mildew of cereals and grasses is recorded every year, and its greatest intensity is in south-eastern and south-western regions. The degree of infestation by B. graminis varies every year. This means that the disease occurs every year, in greater or lesser severity. Therefore, it requires monitoring (harmfulness thresholds) and chemical control practically in every vegetation season. Nowadays, an important role is played by immunological breeding. In breeding programs, resistance genes from wild crop forms, primitive and indigenous varieties are an effective tool. The introduction of effective resistance genes into cultivated varieties is a common procedure used in breeding program.<br />The aim of this study was to describe the fungal disease of plants from the group of powdery mildews caused by <br /> B. graminis as an overview.

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Rhabdocline needle cast — most recent findings of the occurrence of Rhabdocline pseudotsugae in Douglas-fir seeds

Botany ◽

10.1139/cjb-2013-0238 ◽

2014 ◽

Vol 92 (6) ◽

pp. 465-469 ◽

Cited By ~ 4

Author(s):

Kristin Morgenstern ◽

Matthias Döring ◽

Doris Krabel

Keyword(s):

North American ◽

Fungal Pathogens ◽

Douglas Fir ◽

Chain Reaction ◽

Disease Symptoms ◽

Needle Cast ◽

Source Of Infection ◽

Potential Source ◽

Fungal Dna ◽

Polymerase Chain

Rhabdocline pseudotsugae Syd. is one of the major fungal pathogens in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). To date, macroscopic and microscopic analyses of disease symptoms have resulted in the description of the Rhabdocline needle cast pathogen as a highly specialized needle parasite. In practice, the parasite has traditionally been identified by its fruiting bodies on infected needles. But nevertheless, it is not possible to detect the fungus if typical morphological traits are absent. Polymerase chain reaction (PCR) based techniques facilitate the detection of even small quantities of fungal DNA, regardless of the existence of visible fungal structures. Recent investigations into paths of infection and distribution using PCR have identified R. pseudotsugae in various types of Douglas-fir tissue including embryonic tissue which did not exhibit any symptoms. Taking these findings as a basis, the present study systematically tested seeds from five German, 13 North American, and two Ukrainian areas of origin for infection with R. pseudotsugae. The fungus was definitively detected in samples from five German and seven North American areas of origin. Nineteen percent of the tested seeds were infected. This indicates that infected seeds might represent another potential source of infection in addition to the ascospore-based distribution of R. pseudotsugae.

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Microscopy studies of powdery mildew on summer squash

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008691x ◽

1991 ◽

Vol 49 ◽

pp. 520-521

Author(s):

John S. Gardner ◽

W. M. Hess

Keyword(s):

Powdery Mildew ◽

Tip Growth ◽

Hyphal Growth ◽

Plant Tissues ◽

Vegetative Hypha ◽

Powdery Mildews ◽

Summer Squash ◽

Conidial Formation ◽

Polar Tip Growth ◽

Short Conidiophore

Powdery mildews are characterized by the appearance of spots or patches of a white to grayish, powdery, mildewy growth on plant tissues, entire leaves or other organs. Ervsiphe cichoracearum, the powdery mildew of cucurbits is among the most serious parasites, and the most common. The conidia are formed similar to the process described for Ervsiphe graminis by Cole and Samson. Theconidial chains mature basipetally from a short, conidiophore mother-cell at the base of the fertile hypha which arises holoblastically from the conidiophore. During early development it probably elongates by polar-tip growth like a vegetative hypha. A septum forms just above the conidiophore apex. Additional septa develop in acropetal succession. However, the conidia of E. cichoracearum are more doliform than condia from E. graminis. The purpose of these investigations was to use scanning electron microscopy (SEM) to demonstrate the nature of hyphal growth and conidial formation of E. cichoracearum on field-grown squash leaves.

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Incidence of powdery mildew on cucurbit plants in Katsina, Nigeria

Journal of Phytology ◽

10.25081/jp.2019.v11.20190130 ◽

2019 ◽

pp. 05-09

Keyword(s):

Powdery Mildew ◽

Low Cost ◽

Frequency Of Occurrence ◽

Sphaerotheca Fuliginea ◽

Causal Organism ◽

Powdery Mildews ◽

Powdery Mildew Disease ◽

Important Disease

The presence study deals with powdery mildews in various cucurbits in Katsina city (Barhim Estate, Kofar Durbi, Kofar Sauri, Kofar Marusa and Low Cost), Nigeria. The finding shows that the areas infested with powdery mildew is one of the important disease of cucurbits. The Sphaerotheca fuliginea was identified to be the causal organism present on all observed cucurbits in the study. Highest frequency of disease was found in Kofar Sauri(79%) fallowed by Kofar Marusa (68%), Kofar Durbi (66%), Barhim Estate (65%) and the lowest frequency of occurrence of disease was found in Low Cost (55%).The intensity of the disease was moderate to severe in general but it was high in many fields, the area-wise variation was also noticed. On vegetables, the highest frequency of occurrence of powdery mildew disease was observed on L. cylindrica (76.4%) followed by C. moschata (60%), C. sativus (59.3%), C. vulgaris (53.9%) and lowest was found on C. melo (44.4%). The highest intensity of disease was found on C. moschata, followed by L. cylindrica, C. sativus, C. vulgaris and C. melo.

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PCR-detectable Candida DNA exists a short period in the blood of systemic candidiasis murine model

Open Life Sciences ◽

10.1515/biol-2020-0075 ◽

2020 ◽

Vol 15 (1) ◽

pp. 677-682

Author(s):

Zheng-Xin He ◽

Hui-Hai Zhao ◽

Fu-Kun Wang

Keyword(s):

Heat Shock ◽

Murine Model ◽

Fungal Pathogens ◽

Clinical Medicine ◽

Diagnostic Techniques ◽

Systemic Candidiasis ◽

Blood Samples ◽

Short Period ◽

Polymerase Chain ◽

Medicine Today

AbstractInvasive candidiasis is a major challenge to clinical medicine today. However, traditional fungal diagnostic techniques and empirical treatments have shown great limitations. Although efforts are necessarily needed in methodology standardization and multicenter validation, polymerase chain reaction (PCR) is a very promising assay in detecting fungal pathogens. Using a “heat-shock” DNA preparation method, a rapid and simple PCR protocol for quantification of the Candida albicans (C. albicans) ribosomal DNA was established. The PCR assay could detect Candida DNA as low as 10 CFU/mL in samples prepared by the heat-shock protocol, without any cross-reaction with DNA prepared from other Candida spp. and bacterial pathogens. For simulated blood samples, the PCR test sensitivity of whole blood samples was better than that of plasma and blood cells. In the systemic candidiasis murine model, detectable DNA was only observed within 24 h after C. albicans SC5314 injection, which is much shorter than that observed in the kidney.

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