Genetic variations among the isolates of <i>Bipolaris Maydis</i> based on phenotypic and molecular markers

Nadeem, A.; Hussain, S.; Fareed, A.; Fahim, M.; Iqbal, T.; Ahmad, Z.; Saeedullah,; Karim, R.; Akbar, A.

doi:10.1590/1519-6984.253147

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Original Article • Braz. J. Biol. 84 • 2024 • https://doi.org/10.1590/1519-6984.253147 linkcopy

Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Variações genéticas entre os isolados de Bipolaris Maydis podem ser baseadas em marcadores fenotípicos e moleculares

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Keywords: Bipolaris maydis; genetic variations; ITS; maydis leaf blight; RAPD

Isolates	GenBank Accession Numbers	Sites	District	Elevation (m)	N	E
Bm1	MN783607	Warsak	Peshawar	382	33^o57.534	071^o 31.082
Bm2	MN783608	Pandu	Peshawar	316	33^o 59.716	071^o 36.523
Bm3	MN783609	Shahbaz gari	Mardan	305	34^o 13.580	072^o 09.800
Bm4	MN783610	Bhala gari	Mardan	307	34^o 13.588	072^o 07.295
Bm5	MN783611	Azakhel	Nowshera	285	34^o 00.161	071^o 52.850
Bm6	MN783612	Pirsabak	Nowshera	288	34^o 03.447	072 ^o 01.661
Bm7	MN783613	Shoulgara	Charsadda	287	34^o 08.799	071^o 42.752
Bm8	MN783614	Shahmansoor	Swabi	314	34^o 04.072	072^o 26.557
Bm9	MN783615	Central swabi	Swabi	328	34^o 06.275	072^o 27..882
Bm10	MN783616	Central charsadda	Charsadda	293	34^o 09.688	071^o 44.932
Bm11	MN783617	Nesata	Charsadda	302	34^o 11.049	071^o 46.804
Bm12	MN783618	Dargai	Charsadda	304	34^o 10.599	071^o 52.114
Bm13	MN783619	Laachi	Kohat	610	33^o364.0608	071^o 2641.2
Bm14	MN783620	Mung	Haripur	588	33^o 59.9982	072^o 56.0454
Bm15	MN783621	Khan pur	Haripur	550	33^o 10.168	072^o 13.221

Primer	Sequence	Product size (bp)	Annealing temperature (^oC)
OPF-1	ACGGATCCTG	10	34.2
OPI-2	GGAGGAGAGG	10	34.7
OPI-7	CAGCGACAAG	10	34.4
OPI-9	TGGAGAGCAG	10	34.0
OPL-8	AGCAGGTGGA	10	36.9
OPF-9	CCAAGCTTCC	10	33.2
OPA-1	CAGGCCCTTC	10	37.8
OPA-3	AGTCAGCCAC	10	35.2

Isolates	Radial growth (cm)	Conidial septation	Conidial conc ml^-1 (thousand)	Aggressiveness			Fungicide sensitivity against Carbendazim
Isolates	Radial growth (cm)	Conidial septation	Conidial conc ml^-1 (thousand)	Disease severity	Virulence pattern		Colony diameter	% growth allowed in comparison to control (%)
Bm1	2.51 d	3	86.66 bc	3.05 abc		HV	2.41 abcd	53.55
Bm2	6.10 a	2	80.00 c	2.20 fgh		MV	2.0 bcde	44.44
Bm3	3.60 cd	3	73.33 c	1.95 h		LV	2.0 bcde	44.44
Bm4	4.00 bcd	5	80.00 c	2.62 bcdef		MV	2.38 abcd	52.88
Bm5	2.73 d	3	73.33 c	3.22 a		HV	2.38 abcd	52.88
Bm6	3.16 cd	2	80.00 c	3.17 ab		HV	1.78 cde	39.55
Bm7	5.33 ab	4	86.66 bc	2.5 cdefgh		MV	2.48 abc	55.11
Bm8	6.23 a	4	43.33 d	2.05 gh		MV	3.00 a	66.66
Bm9	3.73 cd	4	51.66 d	2.42 efgh		MV	2.71 ab	60.22
Bm10	4.44 bc	3	76.66 c	2.55 cdefg		MV	2.10 bcde	46.66
Bm11	2.66 d	4	110.33 a	2.87 abcde		MV	1.78 cde	39.55
Bm12	5.33 ab	3	46.66 d	3.02 abcd		HV	1.71 de	38.00
Bm13	3.08 cd	5	96.66 b	2.22 fgh		MV	2.01 bcde	44.66
Bm14	2.81 d	4	86.66 bc	2.17 fgh		MV	2.95 a	65.55
Bm15	3.58 cd	4	36.66 d	2.47 defg		MV	1.58 e	35.11

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Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
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[1] LV: Least Virulent with disease severity = ≤ 2.0; MV: Moderately Virulent with disease severity= 2.1-2.9; HV: Highly Virulent with disease severity = 3.0-5.0. Known standard (control) for fungicide sensitivity: 5.

[2] Means followed by the different letters (abcdefgh) in the same column are significantly different from one another according to least significant difference (LSD) test