Research Article

Erysiphe diffusa, a New Record for Powdery Mildew Fungi from Pakistan Breeding

Afshan Akbar1, Saliha Afzal2*, Najam Ul Sehar Afshan2, Muhammad Fiaz1 and Abdul Nasir Khalid2

1Department of Botany, Hazara University, Mansehra, Khyber Pakhtunkhwa, Pakistan; 2Institute of Botany, University of the Punjab, Lahore, Pakistan.

Received | December 22, 2023; Accepted | May 11, 2024; Published | June 20, 2024

*Correspondence | Saliha Afzal, Institute of Botany, University of the Punjab, Lahore; Email: [email protected]

Citation | Akbar, A., S. Afzal, N.S. Afshan, M. Fiaz and A.N. Khalid. 2024. Erysiphe diffusa, a new record for powdery mildew fungi from Pakistan breeding. Biologia (Lahore), 70(1): 16-20.

DOI | https://dx.doi.org/10.17582/journal.Biologia/2024/70.1.16.20

Keywords | Ascomycota, Erysiphales, ITS, Pathogenic fungi, Phylogeny

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

A group of infective fungi belonging to Erysiphaceae, commonly known as powdery mildews, are causing a serious disorder on approximately 10,000 angiosperm plants species (Bradshaw and Tobin, 2020; Amano, 1986). From Pakistan, only 50 species in 10 genera of Erysiphaceae are confirmed. Erysiphe R. Hedw. is the biggest genus of Erysiphaceae with twenty-two species in Pakistan, (Afshan et al., 2021; 2022, 2023). Previously, two species of Erysiphe i.e., E. communis (Wallr.) Schltdl. and E. glycines var. Lespedezae (Braun and Zheng, 1985) were observed on the plant genus Lespedeza Mich., worldwide (Manjunatha et al., 2020; Far and Rossman, 2012). Genus Lespedeza comprises about 40 species, found in East and Tropical Asia, Tropical Australia and Temperate North America. Five species of Lespedeza occur in Pakistan. During field scrutiny in Khyber Pakhtunkhwa, plants of Lespedeza with a white powdery mildew fungus on the leaves were found in Swat, and Battagram districts. They were collected, dried and labelled.

Microscopic and molecular phylogenetic investigation of parasitized plants indicate that the causing agent of the disease is associated with Erysiphe. A thorough explanation is provided including illustrations of morpho-anatomical structures and a phylogram to demonstrate its accurate taxonomic position with the genus Erysiphe.

Materials and Methods

Study area

During field visits of district Swat and Battagram (Khyber Pakhtunkhwa) of Pakistan in 2021, samples of Lespedeza tomentosa (Thunb.) Siebold ex Maxim. infected with powdery mildews were gathered. Swat is located in 34.80°N; 72.36°E and 980 meters above sea level. Biari is a small town sited at 34.38°N, 73.24°E at height of 1904 m. a.s.l. in district Battagram. The climatic zone varies from moist temperate to sub-tropical regions (Haq et al., 2010; Haq, 2012). A study of powdery mildew fungi on foliage and inflorescence of Lespedeza tomentosa was carried out.

Collection and preservation

The samples of diseased plant were shadow dehydrated on blotting sheets. They were preserved by pressing and placing them in airtight polyethylene envelops, in brownish paper wrappers with collection notes. The samples were placed at the herbarium of Department of Botany, Hazara University, Mansehra (HUP) and at the Institute of Botany, University of the Punjab, Lahore (LAH).

Morphological description

Photographing infected plant samples was conducted in the field later with EMZ-TR stereomicroscope (Meijii Techno, Japan). The fungi on plants were mounted with lactic acid and examined with with LABOMED light microscope. Measurements of microscopic structures i.e., chasmothecia, appendages, asci, ascospores, conidiophores, conidia and appressoria were captured with Scope Image camera software.

Molecular analysis

Molecular phylogenetic analysis was executed. Gene JET Plant Genomic DNA Purification Mini Kit #K0791 (Thermo Fisher Scientific, Waltham, USA) was used to isolate the genomic DNA according to the manufacturer’s guidelines. The rDNA Polymerase Chain Reaction was used to amplify PMITS1 and PMITS2 for internal transcribed spacer regions as forward and backward primers (Cunnington et al., 2003). The sequencing were done by TsingKe, China. BioEdit software v.7.2.5 was used for consensus sequences from both primers reads (Hall, 1999). Online NCBI BLAST (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) was intended for seeking and gathering similar sequences of ITS regions from GenBank database (NCBI, 2022). The maximum percentage of uniqueness and query analysis of sequences by linked taxa was recorded. MAFFT (Multiple sequence alignment tool) were used to align all sequences together with the original sequences (Kattoh et al., 2019). The software MEGA 7.0 was used to develop a phylogenetic tree via Maximum Likelihood with 1000 rapid bootstrap copies, using Kimura 2-parameter (Tamura et al., 2013).

Results and Discussion

Morphological study

Erysiphe diffusa (Cooke and Peck) U. Braun and S. Takam. = Erysiphe lespedezae (Zheng and Braun, 1983) = Erysiphe glycines var. lespedezae (Zheng and Braun, 1985), Figures 1 and 2.

 

Mycelium amphigenous, effuse, seldom in spots, transient to persistent; Conidiophores erect, straight to curved, sometimes flexuous, foot cells cylindrical, 25–38 × 8–10 μm, having 1–2 shorter cells that form conidia singly–Conidia were ellipsoid–cylindrical (–doliiform), 25–35 × 11–17 μm. Chasmothecia sporadic to gregarious, (65) 111–119 μm in diam., sometimes larger; Peridium cells unevenly polygonal, 10–22(–30) μm in diam.; appendages 7–30, arising equatorially, short appendages stiff, about 6–11 μm wide, ellipsoid–obovoid, saccate–Asci 4–10, 40–71 × 26–48(–50) μm, short to long stalked, 3–6 spored–Ascospores ellipsoid–ovoid, 15–21 × 9–15 μm, colourless.

 

Material examined

On Lespedeza tomentosa (Papilionaceae), with anamorphic and teleomorphic stages, Pakistan, Khyber Pakhtunkhwa, District Swat, 980 m a.s.l., 17 August, 2021, (Afzal et al., 2023)(PM-74) (LAH-38040); On L. tomentosa, with teleomorph stage, Pakistan, Khyber Pakhtunkhwa, Hazara Division, Battagram District, Biari, 1604 m. a. s. l., 13 October, 2021, (AFE022) (HUP voucher no. 15645).

Molecular phylogenetic analyses

The ITS locus was amplified for all specimens. For reconstruction of frontward and backward sequences, the BioEdit sequence alignment editor was used (Hall, 1999). For a subsequent phylogenetic evaluation, the closest matching sequences were copied from GenBank. Multiple sequence alignment was using MAFFT v 7.0 with all restrictions fixed to default standards (Katoh et al., 2019).

 

Thirty ITS sequences were analysed, of which twenty-eight were already in GenBank (Xiao et al., 2020). A phylogenetic tree was developed from a data matrix of 700 related nucleotide sequences, which consisted for 397 conserved 196 variables, 97 parsimony-informative, and 98 singletons variants. Bulbomicroidium bauhiniicola (Braun and Cook, 2012) was picked as an outgroup. In phylogram (Figure 3), the sequences of fungi on L. tomentosa, grouped with E. diffusa with 100% similarity with other sequences (MT471987, MW290429, MT471984, KP794929 and KP794928) from Taiwan and China.

Lespedeza tomentosa is a shrub belonging to the family Papilionaceae that occur in some countries of East Asia such as China, Korea, Japan, and Pakistan. From August to October, 2021, sporadic white to cottony colonies were monitored on Lespedeza foliage that spread to stems producing a minor chlorosis in Swat and Battagram districts, Khyber Pakhtunkhwa, Pakistan. Morphologically, the conidiophores and conidia of our specimens (PM-74, AFE022) were similar to those of Erysiphe diffusa (= Erysiphe lespedezae). The fungus was determined to be E. diffusa based on its mopholocial features and ITS rDNA sequences with 100% similarity with other sequences of E. diffusa from Taiwan and China (MT471987, MW290429, MT471984, KP794929 and KP794928) from Taiwan and China. E. diffusa as E. lespedezae on Lespedeza tomentosa has been reported from China and in Asia (Braun and Cook, 2012; Farr and Rosman, 2017), as well as in Japan. In Korea, E. diffusa (= E. pisi DC ex. Saint Amans), has been testified on Lespedeza bicolor Turcz. and L. cyrtobotrya Miq. (Shin, 2000). E. diffusa on Bauhinia purpurea L. was described from Thailand by Meeboon and Takamatsu (2017), on Quercus species in China and Russia (Lee, 2013). Hence, Erysiphe diffusa on Lespedeza tomentosa is new record from Pakistan.

Conclusions and Recommendations

Morphological observations and ITS rDNA sequences confirm the identity of powdery mildew isolate of Lespedeza tomentosa in Pakistan as Erysiphe diffusa. This study reports the first record of this fungus in Pakistan.

Acknowledgments

We are grateful to Dr. Seyed Akbar Khodaparast, Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Iran for his help in pathogen identification and also grateful to Prof. Dr. Abdul Rehman Niazi, Institute of Botany, University of the Punjab, Lahore, Dr. Jan Alam (Associate Professor) and Dr. Abdul Majid (Assistant Professor), Department of Botany, Hazara University Mansehra, for their help in the field work.

Novelty Statement

This study presents the first documented occurrence of Erysiphe diffusa, a powdery mildew fungus, on Lespedeza tomontosa. This novel host-pathogen interaction expands our understanding of the fungal pathogen’s host range and provides valuable insights into its ecological significance.

Key novelties

• New host record: Erysiphe diffusa has not been previously reported to infect Lespedeza tomontosa.
• Ecological implications: This discovery contributes to a better understanding of the fungal pathogen’s distri-bution, host specificity, and potential impact on the health and growth of Lespedeza tomontosa.
• Disease management: The identification of this new host-pathogen interaction may inform future disease management strategies for Lespedeza tomontosa, particularly in regions where Erysiphe diffusa is prevalent.

Author’s Contribution

Afshan Akbar: Conceptualization, data curation, writing and review.

Saliha Afzal: Conceptualization, data curation, writing and review.

Najam Ul Sehar Afshan: Conceptualization, administration, review and writing.

Muhammad Fiaz: Administration. Abdul Nasir Khalid: Administration, review and writing.

Conflict of interest

The authors have declared no conflict of interest.

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