Screening of Lentil Fields for Presence of Fusarium Wilt and Root Rot in Türkiye under Terrestrial Climate

Summary

A study in Yozgat province found that biotic factors, such as fungal diseases like wilt and root rot, limit lentil production. Fusarium isolates were found in 95.4% of samples, suggesting periodic local surveys and regular monitoring for sustainable technology development and effective control strategies.

Abstract

Lentil is an important self-pollinated legume crop plant. Its production is limited by various biotic factors, especially fungal agents causing the wilt and root rot complex. The study aimed to understand the regional epidemiology and etiology of phytopathogenic fungal agents to develop control strategies against soilborne Fusarium spp. This study investigated 83 lentil sowing localities in Yozgat province for wilt, root and crown rot diseases caused by common Fusarium species during 2022 and 2023. Symptomatic lentil plants were collected for fungal isolation and identification. The Fusarium isolates were grouped according to colony morphology and cultured on PDA medium. Moreover, genomic DNAs obtained from Fusarium isolates were analyzed using PCR and compared with other Fusarium isolates registered in the NCBI GenBank. Genetic relationships among Fusarium isolates were determined using the Maximum Parsimony (MP) method in the Mega 11 program. The results, mean incidence and disease severity rate of wilt and root rot diseases in Yozgat province were determined to be 16.9% and 38.6%, respectively. Fusarium isolates were found in 95.4% of the samples. There was 99.5% to 100% nucleotide sequence homogeneity among F. oxysporum, F. culmorum, F. graminearum, F. acuminatum and F. solani isolates, and the most isolated species was F. oxysporum. The MP dendrogram of Fusarium isolates was divided into two main branches, the first branch included all F. solani isolates. The second main branch included other Fusarium species isolated in the present study and in NCBI GenBank. The study suggests periodic local surveys to determine the frequency of Fusarium wilt for suppression in lentils. Timely suppression of Fusarium-based damages is strongly suggested to control the disease and conserve the lentil production system.

Introduction

Lentil (Lens culinaris Medik.), a small edible grain legume belonging to the Fabaceae family, is a self-pollinating, cool-season crop with needle-like leaves and white to pale purple or dark purple flowers¹. It was domesticated by humans about 10,000 years ago in the Mesopotamian part of the Fertile Crescent and quickly spread to the New World, including the Mediterranean Basin and Central Asia, and later it was naturalized to the Americas². The world lentil cultivation area is about 5.5 million hectares with production of 6.6 million tons³. Türkiye ranks 4^th in l

Protocol

NOTE: The details of the reagents and the equipment used in the study are listed in the Table of Materials.

1. Field survey, sampling, and fungal isolation

NOTE: Survey work was carried out in 2022 and 2023, according to Endes¹⁶. A total of 83 lentil planting areas covering nine districts in Yozgat province were observed for wilt, root, and root collar rot disease (Figure 1).

1. Select lentil fields with over 1000 m² area as a sampling area. Collect each sample by walking randomly from the border to the center or middle of

Results

Determination of disease parameters
A total of 83 lentil sowing areas covering nine different regions of Yozgat were evaluated in terms of wilt, root, and crown rot disease symptoms were surveyed, extending over an area of 1.1984 x 10⁶ m² (Table 2). Wilt or root rot disease symptoms were encountered in all fields. However, the incidence of wilt and root rot disease in Yozgat was determined as 16.9%, with disease severity of 38....

Discussion

Fusarium wilt is known to cause serious economic yield losses in some parts of the world³¹. The disease was first reported in Hungary³² and later reported in many countries such as Egypt, India, Myanmar, Nepal, Pakistan, Türkiye, Syria, and the USA³³. Kumar et al.³⁴ reported a wide distribution of lentil wilt, root, and root collar rot with reports of occurrence in at least 26 countries worldwide. In a recent stu...

Materials

Name	Company	Catalog Number	Comments
(S)-lactic acid	Merck	100366	Was used as an antibiotic in studies.
2-propanol	Merck	109634	Used in molecular studies.
Adjustable micro automatic pipette (0.1-2.5 µL)	Eppendorf Research	LB.EP.3123000012	Used to measure small volumes of liquids.
Adjustable micro automatic pipette set (2 – 20 µl, 20 – 200 µl, 100 – 1.000 µl)	Eppendorf Research	LB.EP.4924000916	Used to measure small volumes of liquids.
Agar	Merck	110453	For use in making fungal media.
Agarose	Sigma-Aldrich	18300012	For use in gel preparation in electrophoresis.
Air conditioning room	İklimlab		Was used to grow plants under controlled conditions.
Ampisilin	Sigma-Aldrich	A9393	Was used as an antibiotic in studies.
Analytical precision balance	Shimadzu ATX224		Was used to weigh the solid materials used in the study.
Autoclave sterilizer	Zealway	GF-120DR	It was used to sterilize solid and liquid materials at every stage of the study.
Binocular microscope	Leica DM750		For use in morphological diagnosis.
Biological safety cabinet	HFsafe Class II A2		To ensure the safety of the work area, the user, the environment and the operation.
Centrifugal	DLAB DM1424	LB.DL.903001124	Used to separate particles in a sample based on their shape, size and density
Chloramphenicol	Sigma-Aldrich	220551	Was used as an antibiotic in studies.
Cork-borer set	Sigma-Aldrich	Z165220	It was used to take samples from fungus culture in petri dishes.
Cover glass and slide	ISOLAB	075.01.006 / 075.02.005	Was used in the preparation process for microscope studies.
D(+)-glucose monohydrate	Merck	108342	For use in making fungal media.
DFC450 with digital camera	Leica		Digital microscope camera with c-mount interface and with a 5 megapixel ccd sensor.
Dm750 binocular microscope	Leica	MIC5246	Was used for morphological identification of fungi.
DNA gel electrophoresis	thermo fisher scientific	B2-UVT
Dna gel loading dye (6x)	Thermo Scientific	R0611	For use in molecular diagnostics.
dNTP mix	Thermo Scientific	R0192	Used in molecular studies.
Dreamtaq pcr master mixes (2x)	Thermo Scientific	K1082	For use in molecular diagnostics.
Drigalski spatule	ISOLAB	082.03.001	It was used to scrape and spread fungal cultures grown in petri dishes.
Edta	Thermo Scientific	17892	For use in molecular diagnostics.
Ethanol	Merck	100983	Used in molecular studies and surface disinfection studies.
Ethidium bromide	Sigma-Aldrich	E7637	Used to stain dna in gels during gel electrophoresis.
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E6758	Used in molecular studies.
Filter paper	ISOLAB	107.58.158	Used in stock culture studies.
Forced air drying cabinet	ZHICHENG ZXDS-A-1090		For use in incubation processes.
Fume hood	Elektromag EM1201	LB.EM.EM1201	It was used to control harmful chemical vapors, gases and dust.
Gel imaging system	Syngene G:BOX Chemi XX6		For use in molecular diagnostics.
Generuler 50 bp dna ladder	Thermo Scientific	SM0372	For use in molecular diagnostics.
Glacial acetic acid	Merck	1005706	Used in molecular studies.
Glycerol	Merck	104094	For use in stock culture of fungi.
Lancet	ISOLAB	048.50.002	Used to remove diseased tissue from plant samples.
Magnesium chloride	Sigma-Aldrich	814733	Used in molecular studies.
Measuring tape	ISOLAB	016.07.500	Used to measure liquid volumes.
Microcentrifuge tubes	ISOLAB	0778.03.001 / 0778.03.002 / 0778.03.003	Used to store different volumes of liquids.
PCR tube	ISOLAB	123.01.002	It was used to put dna mix in pcr studies.
Petri dishes	ISOLAB	120.13.090	For use in growing fungus culture.
Pipette tips	ISOLAB	005.01.001 / 005.01.002 / 005.01.003 / 005.01.004	To transfer liquid volumes used in analyses.
Plastic bag	ISOLAB	039.30.005	Was used to transport samples to the laboratory.
Plastic pot	ToXA		Was used for growing plants.
Pliers, clamps	ISOLAB	048.08.130	It was used to put filter papers into envelopes after the fungus grew in the petri dish.
Porcelain mortar	ISOLAB	038.02.150	Was used to crush fungal mycelia.
Potato dextrose agar	Condalab	1022	For the identification and cultivation and of fungi.
Pure water system	human CORPORATION	LT.HC.NHP009	Was used in solution preparation and analysis throughout the studies.
Refrigerator (+4 °C / -20 °C)	Vestel		For use in the storage of stock materials.
Rifampicin	Sigma-Aldrich	557303	Was used as an antibiotic in studies.
Sodium acetate	Merck	106268	Used in molecular studies.
Sodium chloride	Merck	1064041000	Used in molecular studies.
Sodium dodecyl sulfate	Sigma-Aldrich	436143	Used in molecular studies.
Sodium hypochlorite solution	Merck	105614	Used for surface disinfection.
Spatula	ISOLAB	047.33.210	It was used to scrape the fungus culture growing in petri dishes.
Streptomycın sulfate	BioShop Canada	STP101	To prevent contamination in fungal culture cultivation.
Teksoll extra pure	Tekkim	TK.200650	For use as a disinfectant in all stages of work.
Tetrasiklin	Sigma-Aldrich	T3258	Was used as an antibiotic in studies.
Thermal cycler PCR	Bio‐Rad T100		For use in genomic analyses.
Thoma lam	ISOLAB	075.03.002	For use in spore counting.
Tris HCL	Roche	10812846001	Used in molecular studies.
Trizma	Sigma-Aldrich	T1503	Used in molecular studies.
Tween 80	Merck	822187	For use in spore solution in pathogenicity testing.
Vortex mixer vorteks	Velp WIZARD	LB.VLP.F202A0175	Used to mix substances in liquid volumes.
Water baths	Memmert WNB 22	1018-5702	It was used during incubation in dna extraction studies.