Vegetative compatibility and rep-PCR DNA fingerprinting groups of Fusarium solani isolates obtained from different hosts and their pathogenicity

Document Type: Original Article

Authors

1 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

2 Plant Protection Research Department, Agricultural and Natural Resources Research Center of Khorasan-Razavi province, Mashhad, Iran

Abstract

Fusarium solain is the most important pathogen of huge range of plant hosts, especially potato in the word, which causes tuber rot in storage and root rot of potato plants in fields. Fifty four isolates from potato, bean, chickpea and cucurbit (melon, watermelon and cucumber) was subjected in a study through analysis of vegetative compatibility groups (VCGs) and rep-PCR DNA fingerprinting. Nit mutants were used to force heterokaryon formation to determine VCGs and Twenty three groups were determined which designated as VCG A to VCG W. VCG A, was the largest group with 18 members and VCG B, VCG C and VCG D were composed of 8, 6 and 3, respectively. Other groups were identified as two or single-member VCGs. Presence of high single-member VCGs indicates that there is a high level of genetic diversity among isolates and isolates of each host classify in different VCGs. Dendrogram generated using data of rep-PCR, suggests high level of genetic diversity among the isolates and no correlation between DNA fingerprinting groups and host and geographical origin of the isolates. Pathogenicity of twenty three F. solani isolates as VCGs representatives originated from mentioned hosts was examined on plants and tubers of Agria cultivar of potato. Except four and two isolates, other isolates were pathogenic on potato plants and tubers. Pathogenicity tests distinguished that F. solani isolates do not have host specific behavior and isolates obtained from non-potato hosts are able to cause disease on potato plants and tubers.

Keywords

Main Subjects


INTRODUCTION

 

Fusarium solani (Mart.) Sacc. is one of the most frequently isolated fungi from soil and plant debris (Zhang et al. 2006). This species comprises phytopat-
hogenic and saprophytic strains. Phytopathogenic strains are grouped in formae speciales based on their host specificity (Snyder & Hansen 1941, Sakurai & Matuo 1961, Roy 1997).The mentioned pathogen is one of the major causes of potato disease in Iran, but unfortunately, the specific association between the isolates of F. solani and potato plants has not been approved and no special host has  been established for them (Moradzadeh Eskandari 2010). Specific forms of this fungus are not distinguishable based on the morphological characteristics (Sugaet al. 2002). The separation and identification of specific forms and assessment of races diversity within a specific form, solely based on the pathogenicity tests is not accurate. These tests are often influenced by environmental variables, such as temperature, host age and inocula-
tion methods (Correll 1991).

Due to the soil born nature and long-term survival of chlamydospores of this fungus in the soil (Hooker 1981), crop rotation strategy to control the disease cannot be effective. One of the primary goals of plant pathology has been to formulate strategies for disease control. These methods usually have been based on genetic resistance in the host plant or the application of chemical fungicides. Understanding of genetic diversity of fungi is very important to select the best Strategies (Leslie 1993).

Access to the information about the pathogenic variability and genetic transmission potential can be beneficial for researchers to understand the relationships between fungal species and resistance phenotype in breeding programs (McDonald & Linde 2002).

Hence, researchers have used genetic markers for more precise identification and characterization. Determination of vegetative compatibility groups can be a useful method for the identification of specific forms and the establishment of effective strategies for controlling the disease (McDonald & Linde 2002). Fungal isolates that anastomose and form heterokaryons with one another are considered to be vegetative compatible and assigned to a single vegetative compatible group (VCG). Conversely, isolates that are incapable of anastomosing and therefore fail to establish heterokaryons are referred to as vegetative incompatible isolates (Anagnostakis 1982).

Correlations between VCGs and other characters such as pathogenicity could lead to effectual diagnostics (Leslie 1993). On the other hand, due to the existence of self-incompatible isolates in the fungus populations and inability of vegetative compatibility groups in the expression of genetic similarity between these isolates, using more powerful tools is essential to overcome the existing limitations. Thus, the use of molecular markers based on PCR has been considered for this purpose.Repetitive-sequence based polymerase chain reaction (rep-PCR) is based on PCR-mediated amplification of DNA sequences located between specific interspersed repeated sequences in prokaryotic genomes. These repeated elements are termed as BOX, REP and ERIC elements.

The objectives of this study were determination of vegetative compatibility groups to clarify the genetic diversity among F. solani isolates from different hosts and comparison of the pathogenicity of vegetative compatibility groups’ representatives on potato plants in glasshouse and on potato tubers in laboratory to examine the host specificity of the isolates, as well as comparison of vegetative compatibility groups with rep-PCR.

 

MATERIALS AND METHODS

 

Fungal isolates

Fifty four isolates of F. solani including twenty nine isolates from potato (Solanum tuberosum), nine isolates from chickpea (Cicera rietinum), nine isolates from common bean (Phaseolus vulgaris), two isolates from cucumber (Cucumis sativus), three isolates from watermelon (Citrullus lanatus), two isolates from melon (Cucumis melo var. inodorus) collected from different regions of Khorasan-Razavi province in east northern Iran were selected for VCG determination, rep-PCR DNA and pathogenicity tests. 

 

Media

Minimal medium (MM) was prepared by adding 
2 g of NaNOin l L basal medium (Correllet al1987). In an initial test, chlorate-resistant mutants were generated on the medium amended with chlorate (KClO3). In this study, we used three chlorate-containing media, including MMC (MM medium with Chlorate), PDC (Potato Dextrose Agar with Chlorate) and CDAC (Czapek medium with Chlorate). To determine the suitable amount of KClO3, 15, 20, 30, 50 and 70 g/l KClO3 in PDA, MM and CDA was tested. Extremely, 50-70 g/l KClO3 was selected so that MMC was prepared by adding 50-70 g/l KClOto 1 L MM medium, PDC was made by adding 39 g PDA, 5 g Davis agar and 50-70 g/l KCIO3 to 1 L distilled water and CDAC was prepared by adding 
39 g CDA and 50-70 g/l KClOin 1 L distilled water.

 

Generation and characterization of Nit mutants

For each isolate, mycelium was transferred from PDA cultures to the medium containing KClO3. Growth of wild-type strains of F. solani was restricted by chlorate on MMC according to Correll et al. (1986). The plates were incubated at 24 ± 2°C in the dark for 10-14 days. Then, the rapidly expanding sectors growing away from the restricted growth zone were transferred to MM, and those that grew as the thin expansive colonies with no aerial mycelium on MM were considered as nit mutants.

The physiological phenotypes of nit mutants were established by growing them on media containing one of the four different nitrogen sources (nitrate, nitrite, hypoxanthine and ammonium tartrate) following Correll et al(1987) (Table 1).

 

Complementation tests

Complementation tests were conducted on MM. Three 2 mm2 agar blocks containing a Nit M mutant grown on MM were placed equidistantly apart across the center of the Petri dish, and agar blocks of nit 1 or nit 3 mutants grown on MM were placed at the three matching positions, opposing the Nit M blocks in two rows and 1.5 mm on either side of the Nit M blocks. This arrangement provided for complementation between different nit 1 or nit 3 mutants and a single Nit M mutant. Complementation indicating heterokaryon formation was recognized as a line of dense aerial mycelial growth where two nit mutants met together on MM (Correll et al. 1987).

 

Table 1. Identification of nit mutants of Fusarium solani isolates by growing them on different nitrogen sources (Correl et al. 1987).

 

Mutant designation

 

Medium supplement

 

Nitrate

Nitrite

Ammonium

Hypoxanthine

Chlorate

Wild type

+

+

+

+

-

nit 1

-

+

+

+

+

nit 3

-

-

+

+

+

Nit M

-

+

+

-

+

Crn*

+

+

+

+

+

+: Typical wild-type growing, -: Thin growing with no aerial mycelium, Crn*: chlorate resistant utilizing nitrate

 

DNA extraction

Three to four mycelia plugs (each 4 mm in diameter) from PDA cultures were transferred to flasks containing 60 ml of potato dextrose broth, and incubated at room temperature for 6–8 days. Mycelial mass was filtered through a filter paper, washed three times with sterile water, air-dried and then frozen at -80°C and lyophilized prior to use. Lyophilized mycelia were ground in liquid nitrogen into a fine powder with a mortar and pestle. Fungal genomic DNA was extracted using Core-one tm Plant Genomic DNA isolation Kit (Core Bio, Korea) following the manufacturers’ instructions.

 

Rep-PCR analysis

Rep-PCR reaction was performed using BOX primer (1A-1R 5'-CTACGGCAAGGCGACGCTG ACG-3') (McDonald et al. 2000). PCR was carried out using PCR Master Mix kit (CinnaGen PCR Master Kit). Each reaction consisted of the following components: 10 μL of PCR Master Mix (containing 1 U of Taq DNA polymerase, 2 μL PCR buffer, 3 mM MgCl2 and 0.25 mM of each dNTPs), 10 pM primer, 3 μL (30 ng/μl) of fungal genomic DNA and 5.5 μL distilled water in a final reaction volume of 20 μL. PCR was performed in a palm Thermal Cycler (CG1-96, Corbett Research, Australia) with the following PCR program. An initial denaturation step of 5 minutes at 95°C followed by 35 cycles of denaturation at 94°C for 3s and 92°C for 30s, 1 min of annealing at 49°C, extension for 7 min at 72°C and a final extension for 10 min at 72°C.

 

Data analysis

To determine the genetic relationships among the isolates, the presence or absence of DNA bands was converted into binary data (1 for presence and 0 for absence of each band). Similarity matrix was calculated with Dice’s coefficient and the SIMQUAL program of NTSYSpc, ver. 2.1. Cluster analysis was carried out within the SAHN program by using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean).

 

Virulence of VCGs representatives on potato plants in greenhouse

For greenhouse pathogenicity tests, spore suspensions were created following Romberg & Davis (2007). Macroconidia and microconidia were harvested from 2-week-old cultures grown on PDA at 25ºC by adding three ml of sterile water to the plates and scraping the surface of the agar plate with a sterile glass slide. The resulting conidial or mycelial suspension was filtered through eight layers of cheesecloth to remove the mycelial fragments. Conidial concentrations were calculated using a hem cytometer and diluted in water to a concentration of 10macro- and microconidia/ml for pathogenicity tests. The pathogenicity of twenty three F. solani isolates obtained from potatocommon beanchickpea, cucumber, watermelon and melon as VCGs representatives was determined on healthy 8-10 cm-tall potato seedlings of Agria cv., the most frequently potato cultivar in all potato growing regions of Khorasan-Razavi province, Iran. After trimming the roots, the entire root system was immersed in a 10macro- and microconidia/ml suspension of each isolate. Each seedling was transplanted into three separate 15-cm-diameter pots containing sterile soil (5:3:2:1 mixture of farm soil, sand, animal manure and compost). Each treatment was replicated three times. The inoculated plants were maintained in greenhouse at 25ºC to 28ºC for 45- 50 days, and then removed from the pots. Then, they were scored for disease severity based on a 0–3 scale (0 = No disease, 1 = mild root rot, 2 = vascular discoloration of the root and crown, 3 = vascular discoloration of the root, crown and stem) (Moradzadeh Eskandari 2010).

 

Virulence of VCGs representatives on potato tubers under lab conditions

Tubers of Agria cv. were used to determine the virulence of the same VCGs representatives as those used in the pathogenicity tests on plants. Initially, the tubers appearing healthy and uniform in size (100-120 g) were selected and washed to remove excess soil, surface sterilized in 0.5% sodium hypochlorite solution for 10 min, rinsed with three changes of sterile distilled water and then air dried. Afterwards, the tubers were wounded with a four mm-diameter cork borer to a depth of four mm (Theron & Holz 1989) and inoculated with all of the fresh F. solani mycelia (from 1-week-fresh cultures grown on PDA at 25°C) by putting 7 mm PDA blocks. All the wounded potato tubers were wrapped in paper bags (Manici & Cerato 1994) and incubated at 20°C under dark conditions for three weeks. Blocks of PDA medium were used as control. Each treatment was replicated three times. At the end of incubation, tubers were cut through the inoculation points, and the degree of rot was estimated based on a zero to five (or A-F) scale, basically according to Theron and Holz (1989): 0. (A) - Limited discoloration, no exten-
ded dry rot in inoculated areas; 1. (B) - Limited discoloration with the development of dry rot in inoculated areas; 2. (C) - Extensive discoloration with increased dry rot in inoculated areas; 3. (D) - Extensive discoloration with extensive dry rot in inoculated areas; 4. (E) - discoloration and very extensive dry rot, tubers not disappearing completely; 5. (F) - discolora-
tion and very extensive dry rot, tubers disappearing completely.

 

RESULTS

 

Determination of VCGs

Production of chlorate-resistant sectors was very low on MMC, PDC and CDAC containing 15 g/l KC103, but after increasing the concentration of KClO3 in the three media to 50-70 g/l, most of the isolates readily formed chlorate-resistant sectors (Fig. 1a). Totally, fifty two isolates produced chlorate-resistant sectors. These sectors were then transferred to MM containing NaNOas the sole nitrogen source. The sectors with thin expansive growth on MM were considered as nit mutants. A few sectors resistant to chlorate were recovered, but they had wild type colony morphology on MM. Such mutants are known as Chlorate resistant isolates utilizing nitrate (Crn) (Bowden & Leslie 1992).

Four different nitrogen sources were used in order to identify phenotypic classes. Three classes of nit mutants were recovered representing mutations at a nitrate reductase structural locus (nit l, unable to utilize nitrate), a nitrate-assimilation pathway-specific regulatory locus (nit 3, unable to utilize nitrate or nitrite) and loci affecting the assembly of a molybdenum-containing cofactor necessary for nitrate reductase activity (Nit M, unable to utilize nitrate, hypoxanthine or uric acid). The frequency of nit 1, nit 3 and Nit M phenotype was 44.74%, 36.84% and 18.42%, respectively. Nit l mutants were recovered at a higher frequency than nit 3 and Nit M mutants.

Complementation between nit mutants was indicated by the development of a dense aerial growth where the mycelia of the colonies grew together and anastomosed. When Nit M mutants were paired-or were involved in the pairing, complementation occurred more rapidly than those of other nit mutant pairs. When nit 1 and nit 3 mutants were paired, weak vegetative compatibility reactions were obtained. Those isolates that had two mutants available for complementation tests, and had positive intra-strain complementation tests were classified as heterokaryon self-compatible (HSC) and those with negative intra strain complementation tests were considered as heterokaryon self-incompatible (HSI). One of the 52 isolates was HSI. Six multi-strain VCGs were identified among the HSC strains (Fig. 1b). These groups were designated as VCG A to VCG W. VCG A as the largest group had 18 members and VCGs B, C and D comprised of 8, 6 and 3 members, respectively. Each of the VCGs E and F consisted of two members. Seventeen isolates out of 51 HSC isolates were incompatible with all of the other isolates and therefore were considered as single-strain VCGs with designation as VCG G to VCG W. Generally, isolates of each of the VCGs originated from different hosts and usually from different regions (Table 2). VCG A included fourteen isolates of potato, three isolates of chickpea and one isolate of common bean, which belonged to several regions (Chenaran, Sabzevar, Fariman, Torbat-e Heydarieh, Quchan, Nishapur, Mashhad and Birjand). VCG B consisted of seven isolates of potato and one isolate of common bean, obtained from four regions (Chenaran, Fariman, Torbat-e Heydarieh and Mashhad). Three isolates of common bean, one isolate of potato, one isolate of chickpea and one isolate of melon were grouped in VCG C. The members of this VCG were obtained from six regions (Chenaran, Torbat-e Heydarieh, Mashhad, Faruj, Kashmar and Ardabil). VCG D contained 3 isolates of three host plants (potato, common bean and chickpea) and three different regions (Kashmar, Qaen and Torbat-e Heydarieh). Two isolates of chickpea from two different regions (Faruj and Mashhad) were grouped in VCG E. VCG F contained 2 isolate of potato, obtained from Birjand. Therefore, there was no correlation between VCGs and hosts and geographical origin of the isolates (Fig. 1c).

 

Rep-PCR DNA fingerprinting

Totally, 37 fragments were amplified using BOX primer, ranging in size from 2000 to 3500 bp, all of which were polymorphic (Fig. 2). Cluster analysis revealed that all of the isolates examined were placed in two major fingerprinting groups (designated as Foand Fo2) with at least 30% genetic similarity (Fig. 3). Table 3 exhibits diversity of VCGs according to rep-PCR analysis using BOX primer.

Pathogenicity test of VCG representatives on potato tubers revealed that among 23 VCGs representatives tested, 21 isolates showed various virulence and only isolates CH-25 (VCG E) and FW-52 (VCG T) caused no symptoms on tubers of Agria cv. Isolates Fpo-5 (VCG B), Fpo-70 (VCG I) and Fpo-69 (VCG A) from potato possessed the highest virulence (Fig. 5). Details of these tests and isolates grouping are given in table 4.

 

 

Fig.1(a) Production of chlorate-resistant sectors on PDA containing 70 g/l KClOin the isolate FCV-21 of Fusarium solani at 25°C, under dark conditions after 15 days. (b) Heterokaryon formation on minimal medium between nit 1 and Nit M mutants in the isolate C-96 of F. solani at 25°C, under dark conditions after 10 days. (c) Complementation between nit 3 and Nit M mutants of F. solani isolates on minimal medium, and heterokaryon formation.

 

Table 2. Characteristics of nit mutants of Fusarium solani isolates obtained from different host plants in different regions of Khorasan-Razavi province, Iran.

Isolates

Locationa

Year

Host

Phenotype of nit mutants

VCG b

 

 

A

B

C

 

 

2

Quchan

2004

Potato

nit 1

nit 3

-

A

3

Fariman

2004

Potato

nit 1

nit 3

Nit M

J

10

Fariman

2004

Potato

nit 1

nit 3

Nit M

B

12

Nishapur

2004

Potato

nit 1

nit 3

-

A

13

Torbat-e Heydarieh

2004

Potato

nit 1

nit 3

Nit M

A

14

Quchan

2004

Potato

nit 1

nit 3

Nit M

A

16

Quchan

2004

Potato

nit 1

-

Nit M

K

19

Mashhad

2004

Potato

nit 1

nit 3

Nit M

B

20

Torbat-e Heydarieh

2004

Potato

nit 1

nit 3

Nit M

L

F-132

Ardabil

2004

Potato

nit 1

nit 3

-

A

Fpo-1

Chenaran

2007

Potato

nit 1

nit 3

-

C

Fpo-9

Birjand

2007

Potato

nit 1

nit 3

-

A

Fpo-19

Birjand

2007

Potato

nit 1

nit 3

-

A

Fpo-8

Birjand

2007

Potato

nit 1

nit 3

-

A

Fpo-16

Birjand

2007

Potato

nit 1

nit 3

Nit M

F

Fpo-45

Sabzevar

2007

Potato

nit 1

-

Nit M

A

Fpo-44

Chenaran

2007

Potato

nit 1

nit 3

-

A

Fpo-7

Birjand

2007

Potato

nit 1

nit 3

Nit M

B

Fpo-67

Bojnord

2007

Potato

nit 1

nit 3

-

F

Fpo-62

Fariman

2007

Potato

nit 1

nit 3

-

A

Fpo-60

Quchan

2007

Potato

nit 1

nit 3

-

G

Fpo-54

Chenaran

2007

Potato

nit 1

nit 3

-

A

Fpo-69

Esfarayen

2007

Potato

nit 1

nit 3

-

B,A

Fpo-70

Quchan

2007

Potato

nit 1

-

Nit M

A

Fpo-74

Torbat-e Jam

2007

Potato

nit 1

nit 3

-

I

Fpo-76

Fariman

2007

Potato

nit 1

nit 3

-

H

Fpo-87

Fariman

2007

Potato

nit 1

nit 3

-

B

Fpo-22

Qaen

2007

Potato

nit 1

-

-

B,A

Fpo-5

Chenaran

2007

Potato

nit 1

-

Nit M

D

LM-WM

Kashmar

2006

Common Bean

nit 1

nit 3

-

B

LM-13b

Mashhad

2006

Common Bean

nit 1

-

Nit M

C

LM-23

Chenaran

2006

Common Bean

nit 1

-

Nit M

C

LM-25

Torbat-e Jam

2006

Common Bean

nit 1

nit 3

-

B,C

LM-26

Bojnord

2006

Common Bean

nit 1

nit 3

-

V

LM-40

Bojnord

2006

Common Bean

nit 1

nit 3

-

Q

LM-6

Fariman

2006

Common Bean

nit 1

nit 3

Nit M

S

LM-11

Mashhad

2006

Common Bean

nit 1

nit 3

-

R

LM-28

Bojnord

2006

Common Bean

nit 1

nit 3

-

-

CH-2

Torbat-e Jam

2005

Chickpea

nit 1

-

Nit M

D

CH-5

Mashhad

2005

Chickpea

nit 1

-

-

E

CH-7

Fariman

2005

Chickpea

nit 1

nit 3

Nit M

F

CH-14

Mashhad

2005

Chickpea

nit 1

nit 3

-

A

CH-25

Faruj

2005

Chickpea

nit 1

-

Nit M

E

CH-4

Chenaran

2005

Chickpea

nit 1

-

Nit M

A

CH-KH

Khvaf

2005

Chickpea

nit 1

nit 3

Nit M

O

CH-31

Rashtkhvar

2005

Chickpea

-

-

-

-

FCV-21

Khorasan-Razavi

2006

Chickpea

nit 1

nit 3

-

A

C-96

Jajrom

2006

Cucumber

nit 1

-

Nit M

U

C-89

Esfarayen

2006

Cucumber

nit 1

-

-

M

C-102

Kashmar

2006

Melon

nit 1

nit 3

-

C

C-120

Mashhad

2006

Melon

nit 1

nit 3

-

W

FW-52

Fariman

2002

Watermelon

nit 1

nit 3

-

T

FW-54

Nishapur

2002

Watermelon

nit 1

nit 3

-

P

C-67

Jovin

2002

Watermelon

-

nit 3

-

-

                   

Location = different regions of Khorasan-Razavi province, Iran

VCG = vegetative compatibility groups (totally, 54 isolates were grouped in 23 VCGs based on vegetative compatibility tests by pairing nit mutants)

 

Rep-PCR DNA fingerprinting

Totally, 37 fragments were amplified using BOX primer, ranging in size from 2000 to 3500 bp, all of which were polymorphic (Fig. 2). Cluster analysis revealed that all of the isolates examined were placed in two major fingerprinting groups (designated as Foand Fo2) with at least 30% genetic similarity (Fig. 3). Table 3 exhibits diversity of VCGs according to rep-PCR analysis using BOX primer.

 

 

Fig. 2. Electrophoretic patterns on 1.4 % agarose gel of amplified fragments generated from the 21 isolates of Fusarium solani by rep-PCR, using BOX-primer. Lane M is the 1 kb DNA ladder; Lanes1-20; F. solani isolates (16, Fpo-5, Fpo-69, Fpo-9, 
Fpo-74, CH-25, LM-40, 12, Fpo-67, Fpo-87, C-102, 14, FW-52, Fpo-76, LM-23, Fpo-44, Fpo-16, LM-25, C-67, CH-2 and Fpo-8).

 

 

Fig. 3. Dendrogram constructed by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) based on the data of rep-PCR using BOX primer, indicating relationships among Fusarium solani isolates obtained from different host plants. Similarity matrix was produced by Dice`s coefficient. Defined main groups, designated as Fo1 and Fo2 and subgroups of Fo1, designated as Fo1-1 to Fo1-12 are indicated on the right.

 

Table 3. Diversity of Fusarium solani VCG groups according to rep-PCR DNA fingerprinting analysis, using BOX primer.

 

Fingerprinting group

Fingerprinting subgroup

No. of isolates

VCGs

Hosts

Locationa

 

Fo1

Fo1-1

7

VCG A, VCG R, VCG O, VCG S, VCG U

PotatoCommon BeanChickpea, Cucumbers, Watermelon

Mashhad, Khaf, Fariman, Chenaran and Quchan

 

Fo1-2

2

VCG F, VCG P

Potato, Watermelon

Birjandand Nishapur

Fo1-3

7

VCG A, VCG M

PotatoCommon BeanChickpea, Cucumbers

Nishapur, Torbat-e Jam, Mashhad, Jajrom, Esfarayen

Fo1-4

4

VCG J, VCG V, VCG G, VCG C

Potato, Common Bean

Mashhad, Fariman, Bojnord

Fo1-5

6

VCG B, VCG N, VCG L, VCG D, VCG E

PotatoCommon Bean, Chickpea

Mashhad, Fariman, Kashmar, Torbat-e Jam

Fo1-6

2

VCG C, VCG Q

Potato, Common Bean

Bojnord, Ardabil

Fo1-7

3

VCG B, VCG F, VCG W

Potato, Watermelon

Mashhad, Fariman, Mashhad

Fo1-8

7

VCG A, VCGB, VCG K

Potato

Nishapur, Bojnord, Birjand, Chenaran, Fariman, Sabzevar, Qaen

Fo1-9

2

VCG A, VCG D

Potato

Sabzevar, Qaen

Fo1-10

6

VCG A, VCG B, VCG C, VCG T

PotatoCommon Bean, Melons, Watermelon

Kashmar, Fariman, Chenaran, Torbat-e Heydarieh, Bojnord

Fo1-11

1

VCG D

Chickpea

Torbat-e Heydarieh

Fo1-12

8

VCG A, VCG B, VCG H

Chickpea, Potato

Chenaran, Esfarayen, Torbat-e Jam, Birjand

 

Fo2

-

2

VCG A and VCG I

Potato

Qochan

             
a. Sampling regions in Khorasan-Razavi province, Iran. Each region is a town.

 

 

Fig. 4. Pathogenicity of Fusarium solani isolates on potato plants in greenhouse at 25°C. (a) Isolate Fpo-74 obtained from potato (b) Isolate CH-25 obtained from chickpea. In each Fig., the infected plant is on the right side and the healthy plant is on the left side. Photographs were taken 30 days after inoculation.

 

 

Fig. 5. Tuber rot caused by Fusarium solani isolates three weeks after inoculation at 20°C under dark conditions in the lab. (a) No dry rot on potato by the isolate CH-25 obtained from chickpea, with zero disease scale, (b-f) Dry rot on potato by the isolates LM-6, CH-7, Fpo-8, Fpo-69 and Fpo-5 with disease scales of 1, 2, 3, 4 and 5, respectively.

 

Table 4. Pathogenicity of Fusarium solani isolates (VCG representatives) on potato plants in greenhouse and on potato tubers under lab conditions.

Isolates

Host

VCG

Disease index on potato plantsa

Disease severity average on potato tubers b

3

Potato

J

0

1

16

Potato

K

1

0.7

20

Potato

L

0

0.7

Fpo-8

Potato

F

2

2.7

Fpo-62

Potato

G

1

2

Fpo-74

Potato

H

3

2

Fpo-70

Potato

I

3

4.3

Fpo-22

Potato

D

1

2.7

Fpo-69

Potato

A

2

4

Fpo-5

Potato

B

3

4.7

LM-13b

Common Bean

C

0

0.4

LM-40

Common Bean

Q

1

0.7

LM-11

Common Bean

R

0

2.5

LM-6

Common Bean

S

1

0.7

LM-26

Common Bean

V

2

0.7

CH-25

Chickpea

E

1

0

CH-7

Chickpea

N

1

2.5

CH-KH

Chickpea

O

1

2.7

C-89

Cucumbers

M

1

4

C-96

Cucumbers

U

1

2.5

C-120

Melons

W

1

2.5

FW-52

Watermelon

T

1

0

FW-54

Watermelon

P

2

2.5

Disease index value on potato plants obtained from three replicates with 3 plants 45 days post-inoculation, 0 = no disease; 1= mild infection; 2 = severe infection; 3 = 100% infection.

Disease severity average on potato tubers based on Theron & Holz (1987) index (bx) (x = replicate number) obtained from three replicates with 3 tubers 3 week post- inoculation.

 b(b1 + b2 + b 3)/3

 

DISCUSSION

Vegetative compatibility groups (VCGs) have been used to examine diversity and population structure in many fungi. Initially, Puhalla (1985) found that there was a correlation between VCG and forma specialis. Isolates in the same VCGs belonged to the same forma specialis and strains in different formae speciales were grouped in different VCGs (Correll 1991).

Vegetative compatibility has been tested in many fungi using nitrate non-utilizing (nit) mutants to demonstrate heterokaryosis (Leslie 1993). Various factors such as temperature, nutrition and the kind of fungus influence generation of KClO3 resistant sectors in the fungi (Klittich & Leslie 1988). Fusarium solani strains are more tolerant to chlorate than most strains of F. oxysporum and F. moniliform (Correllet al. 1987, Klittich & Leslie 1988). In the present study, most nit mutants were recovered on PDA containing 50-70 g/l KClO3.

Identification of phenotypes of nit mutants showed that nit 1 and Nit M have highest and lowest frequencies, respectively. Frequency of nit mutations in fungi can be influenced by the nitrogen source in medium containing potassium chlorate. Hawthorne et al. (1996) showed that by adding thereonine to the medium containing chlorate, Nit M of the species F. solani increases.

In this study, Nit mutants were obtained for 52 out of 54 isolates of F. solani. One out of 52 isolates was heterokaryon self- incompatible (HIS). Hawthorne et al. (1996) reported that there are many self-incompatible isolates among different isolates of F. solani, but the importance of self-incompatibility in nature is uncertain. Correll et al.(1989) concluded that HSI in Gibberella fujikuroi (Fmonilifome)was under control of a single gene, and Jacobson and Gordon (1990) suggested that it could be a mutant artifact that arises in fungi maintaining for long periods in an artificial culture.

Fifty one heterokaryon self-compatible (HSC) isolates were grouped in 23 VCGs. Seventeen of 51 HSC isolates were incompatible with all the other isolates, and therefore were regarded as single-member VCGs. Presence of high single-member VCGs indicates the high level of genetic diversity among the isolates. Distribution of F. solani isolates obtained from various areas and host plants into different VCGs was observed in the present study. For example, the isolates belonging to VCG A were obtained from potato, common bean and chickpea, and collected from eight regions. VCG D included the isolates from potato, common bean, cucumber and melon, collected from three regions. Besides, only VCG E and VCG F contained the isolates from the same host. VCG E consisted of two isolates from chickpea, and VCG F contained two isolates from potato. Comparison between the isolates from different VCGs and their host origins showed that the isolates from the same host were classified in different VCGs, and had a considerable amount of genetic diversity. Therefore, except for some cases, VCGs grouping in the present study could not separate isolates, especially the potato isolates that had the most frequency in this study based on the host and geographical origin. But in some other studies, VCG grouping could separate isolates of F. solani based on the host origin. For instance, Hawthorn et al. (1996) placed 57 F. solani isolates in 35 VCGs, and reported a direct relationship between VCG and host origin of the isolates. Also, there was no correlation between VCG grouping and geographical origins of the isolates in the present study, except for VCG F with two members that were collected from the same region. On the other hand, the isolates of different geographical areas were placed in the same VCG. This suggests the possibility of distribution of the fungus through infected tubers and agricultural equipment, wind and rain in different regions. According to Elmer (1991), existence of the isolates from different regions in the same VCG, or in the other words, existence of a specific VCG in several areas indicates the selective survival of vegetative compatibility groups. This result was similar to the results of Raouffiet al. (2004) and Mohammadi & Banihashemi (2005). On the other hand, some studies have demonstrated the correlation between vegetative compatibility groups and geographical origins. Rahkhodaei (2000) reported that isolates of F. solani from potato in each VCG were collected from the same region.

Dendrogram generated using data of BOX primers showed that 54 isolates collected from different regions of Khorasan-Razavi province and from different hosts showed >30% similarity. This result suggests a high level of genetic diversity among the isolates. There was no correlation between the identified fingerprinting groups and host and geographical origins of the fungal isolates. These results are in agreement with results from the previous studies on F. solani (Moradzadeh Eskandari 2010, Romberg & Davis 2007, Baghai Raveri et al. 2007). Missing relationships between DNA fingerprinting groups and VCGs observed in this study showed that it could be impossible to separate the isolates based on VCGs using BOX primer. Using VCG grouping and rep-PCR in the present study showed the genetic diversity within F. solani isolates, but both methods were unable to classify the isolates based on the host origin. Therefore, using other methods such as investigation of mating populations will generate further information about the host population of F. solani. Pathogenicity test of twenty three F. solani isolates obtained from different hosts as VCGs representatives on potato plants showed that there was no relation between the host origin and virulence of the isolates, so that two out of four isolates in pathogenicity zero group belonged to potato host. In contrast, non-potato isolates in some cases had more virulence than potato isolates, and were comparable with potato isolates in severity of symptoms. Also, the pathogenicity of twenty three isolates as VCGs representatives on potato tubers showed that except VCG T and VCG E representtatives (from non-potato hosts), the rest of VCGs representatives were able to cause dry rot on potato tubers with diverse virulence.  This result confirmed the results of their pathogenicity on potato plants. In a way, the isolates tested in both pathogenicity tests, regardless of the host origin were grouped in different pathogenicity groups. These results indicated that the pathogenicity of F. solani isolates was not host specific and the isolates obtained from non-potato hosts were able to cause disease on potato plants and tubers. On the other hand, the isolates obtained from potato showed no symptom on potato plants. This result confirmed the results of previous studies (Moradzadeh Eskandari 2010, Romberg & Davis 2007).

 

ACKNOWLEDGMENTS

We gratefully acknowledge the High Council for Research of University of Tehran for financially support and providing materials under grant number 7110024/6/24.

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