Morphological and Molecular Characterization of

VEGETOS

Vol. 28 (1) : 113-121 (2015)

10.5958/2229-4473.2015.00015.4

Morphological and Molecular Characterization of Fusarium spp. causing Post Flowering Stalk Rot of Maize M K Khokhar*, S S Sharma, K S Hooda1 and B L Roat Received: 09.07.2014 / Revised: 27.12.2014 / Accepted: 11.03.2015 / Published online: 30.04.2015 This article is published in open access at www.vegetosindia.org

Abstract Ten isolates of Fusarium spp. (F. proliferatum (4 isolates), F. verticillioides (5 isolates), and F. pallidoroseum (1 isolates) were collected from different parts of Rajasthan from rotted maize stalks and identified by ITCC, IARI (New Delhi, India). The maximum colony diameter (90mm), sporulation (3.4x105 X 3.8x105), size of macroconidia (40.24 X 6.60) and microconidia (10.33 X 1.50) and septation (5-7) were observed in isolate Fv-01 (F. verticillioides) whereas minimum colony diameter (54.70mm), sporulation (0.8 x105 X 1.6 x105), size of macroconidia (17.57 X 1.80) and microconidia (9.09 X 1.48) was observed in isolate Fv-09 (F. proliferatum) . On the basis of cultural and morphological characters of all the ten isolates showed variability, but based on RAPDPCR analysis, these isolates can be categorized into two major clusters I and II at 0.44% similarity coefficient. RAPD-PCR analysis suggested that variability was observed between species of Fusarium recovered from maize and the technique could be use to complement morphological characterization and to determine genetic relationships between the species. Keywords- Maize, PFSR, Fusarium, Pathogenicity, Morphology, RAPD-PCR analysis Introduction Post flowering stalk rot (PFSR) complex play a vital role in affecting the productivity of maize (Zea mays L.) crop in all continents of the world, including USA, Europe, Africa, Asia and Australia (Nur Ain Izzati et al. 2011). This complex is caused by Fusarium verticillioides, Macrophomina phaseolina and Cephalosporium maydis, out of which F. verticillioides; (Saccardo) is of economic importance (Kumar and Shekhar, 2005, Dorn et al. 2009). In India, the disease is prevalent in most of the maize growing areas, particularly in rainfed areas viz., Jammu and Kashmir, Punjab, Haryana, Delhi, Rajasthan, Madhya Pradesh, Uttar Pradesh, Bihar, West Bengal, Andhra

Pradesh, Tamil Nadu and Karnataka (Kaur and Mohan 2012), where water stress occurs after flowering stage of the crop. Reduction in yield due to PFSR disease in susceptible maize germplasm has been estimated to the tune of 39.5 per cent (AICRIP, 2013). The water stress at flowering and high soil temperature help in increasing of the magnitude of the stalk rot symptoms at post flowering stage of maize crop (Smith and McLaren 1997, Khokhar et al. 2014). Most of the commercially grown cultivars have shown a high level of disease incidence at the grain filling stage (Iglesias et al. 2010). Studies on cultural, morphological and molecular variations of predominant pathogen Fusarium spp. are important for germplasm evaluation. Morphological identification of plant pathogenic fungi is the first and the most difficult step in the identification process. This is especially true for Fusarium species. Although morphological observations may not sufficient for complete identification, a great deal of information is usually obtained on the culture at this stage (Rahjoo et al. 2008, Sankar et al. 2011). RAPD-PCR has been successfully used to identify strains and races in phytopathogenic fungi. It has been used for studying inter and intraspecific variability among population from different and same geographic areas (Saharan et al. 2008). The RAPD pattern visualizes variations in total DNA and thus is suitable for differentiating Fusarium spp. isolates. RAPD results obtained in present study enabled fast variability analysis for Fusarium isolates. Traditional markers used to study the variability in plant pathogens are based on the differential hosts, cultural characteristics, morphological markers and biochemical tests. These markers distinguish pathogens on the basis of their physiological characters i.e. pathogenicity and growth behaviour. But these markers are influenced by host age, inoculum quality and environmental conditions. Moreover, these techniques are time consuming, laborious. Differ-

Department of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur-313001, Rajasthan India 1 Indian Institute of Maize Research, Pusa Campus, New Delhi 110 012 *Corresponding author E-mail: [email protected] 113

Morphological and Molecular Characterization of Fusarium Fig 1. Consensus tree showing clustering of Fusarium spp. using RAPD analysis

ential host-pathogen interaction between maize and Fusarium are considered to be non significant. In such cases, molecular markers are used for studying genetic variability in plant pathogens (Sharma et al. 1999). There is little information about Post Flowering Stalk Rot disease in maize has not been extensively studied in this country. Additionally, the causal fungi of PFSR disease had not been molecularly identified until the present study. The aims of this study were (1) to isolate and characterize Fusarium spp. associated with PFSR disease of maize using cultural and morphological methods, and (2) to identify each Fusarium species using RAPD PCR analysis to confirm the results of morphological methods Material and Methods Collection and identification of isolates Maize rotted stem showing typical PFSR symptoms were collected from different locations (Table 1) of Rajasthan during 2012. Isolations were carried out on potato dextrose agar (PDA) plates and subsequently purified by hyphal tip method (Leslie and Summerell 2006). The purified culture was incubated at 28±2°C temperature and used for further studies. The cultures were identified as Fusarium species based on morphological characters with the help of “Laboratory Manual for Identification of Fusarium Species” (Booth 1971) and identification report received from ITCC (Indian Type Culture Collection) IARI New Delhi (Table 1). Cultural and morphological variability The isolates were grown on PDA at 28±2°C temperature for seven days to study the morphological characters like width of mycelium, size of conidia and number of transverse and

longitudinal septa. Data in relation to morphological and cultural characters like, colony diameter, colony color, pigmentation, zonation, topography of the culture, margins of the culture, rate of sporulation, septation and size of macro and micro conidia were studied. Studies of sporulation were also undertaken using the formula given by Pathak (1984). The size of conidia was measured under light microscope at 40X using micrometer. Fifty observations were taken for conidial measurement and mean values were calculated. Molecular variability For the RAPD analysis of pathogen isolates, mycelium of all 10 isolates was grown in potato dextrose broth at 28 ± 2°C for 14 days and was harvested by filtration through filter paper (Whatman No. 1). For DNA isolation 250 mg lypholized mycelium of each isolate was ground in liquid nitrogen in a small sterilized mortar and total fungal genomic DNA was isolated according to CTAB (hexa- decyl tri-methyl ammonium bromide, Sigma chemical Co., St. Louis, USA) method (Doyle and Doyle 1990). Isolated DNA was purified by using 5 µl of each DNA sample on 1% agarose gel to get desired concentration. Initially, 10 mer random primers of OPA, OPG and OPC series of operon technologies were tried for amplifications of two isolates and two primers giving maximum polymorphs were selected for further studies of all isolates. The PCR reaction was set up with a total reaction volume of 25 µl comprised 2 µl (20 ng) fungal genomic DNA, 2.5 µl PCR buffer (10 x) containing MgCl2 (15 mM) 1 µl of 10 mM dNTP, 0.2 µl of Taq DNA polymerase (3 U/ µl), and 2 µl (15 ng) each of 10 mer oligonucleotide primer. The reaction was performed in a thermocycler (Applied Biosystems 114

M K Khokhar et al. Fig 2. Two dimensional principal component analysis showing clustering of Fusarium spp. using RAPD analy-

9700) set to the following: denaturation at 940 C for 2 minutes 45 cycles of 940 C at 1 min. 370 C for 1 min. and 2 min. 720 C for 2 minutes. PCR amplification products were electrophoretically separated on 1% agarose gel stained with EtBr in TAE buffer and photographed under UV light in a Gel Documentation System, (Alpha Innotech Corporation).

Data analysis The amplified fragments were scored as ‘1’ for the presence and ‘0’ for the absence of a band generating the ‘0’ and ‘1’matrix. The data obtained was analysed by obtaining the pair wise genetic similarities using DICE similarity coefficient. Clustering was done using the symmetric matrix of similarity coefficient and cluster obtained based on unweighted pair group arithme-

Table 1: Detailed passport information of Fusarium spp. recovered from samples collected from diseased fields in Maize growing areas of Rajasthan (India). Isolate designation

Fusarium spp.

Location of collection

Land races/ variety

Identification ITCC I.D. No. 9243.13

Fv-01

Fusarium verticillioides

Lohira (Udaipur)

Local land race

Fv-02


Fv-03

Moti composite makka Local land race

9245.13

Fv-04

Fusarium pallidoroseum Fusarium verticillioides

RCA Agronomy field (Udaipur) Banswara Chittorgarh

HM-5

9246.13

Fv-05

Fusarium proliferatum

Menar (Udaipur)

Local land race

9247.13

Fv-06


Dungarpur

Surya

9248.13

Fv-07


Bhilwara

PEHM-2

9249.13

Fv-08


Kota

Aravali Makka

9250.13

Fv-09


Baran

Aravali Makka

9251.13

Fv-10


Dabok (Udaipur)

Pratap makka 3

9252.13

9244.13

115

White Brown Concentric rings No undulation Raised Flat

Violet No undulation Flat

Semi circular (irregular) Circular Circular

Pale yellow No undulation Medium raised Circular 76.40

80.32

54.70 60.22


Fusarium proliferatum Fusarium verticillioides

Fv-08

Fv-09 Fv-10

Fv-07

62.80 71.50 84.65

Fusarium verticillioides Fusarium proliferatum Fusarium proliferatum Fusarium proliferatum Fv-04 Fv-05 Fv-06

Cottony white (superficial) with violet at bottom ring at center Cottony white (superficial) with bluish at bottom center Dirty white cottony Dirty grey white

White Pink Pink No undulation Concentric rings Concentric rings Medium raised Flat Flat

86.44 72.28

Fusarium verticillioides Fusarium pallidoroseum Fv-02 Fv-03

Purple Red at bottom with red cottony (superficial) White grey Purple violet Cottony pink

Semi circular Circular Waivy

Violet Red No undulation No undulation Flat Raised

No undulation Medium raised

Semi circular (irregular) Circular Circular Cottony white


Colony color

Colony diameter 90.00

Fusarium spp.

Isolate designation Fv-01

Table 2. Cultural variability among ten isolates of Fusarium spp. obtained during survey.

Margin

Topography of the culture

Zonation

Pigmentation in culture White

Morphological and Molecular Characterization of Fusarium

tic mean (UPGMA) using sequential agglomerative hierarical nested (SAHN) cluster analysis of NTSYS-PC version 2.0 (Rohlf 1998). Results and Discussion Cultural and morphological variability A total of 10 isolates of Fusarium was obtained from the stems of diseased maize plants. Based on morphological characteristics, 10 isolates were classified as F. verticillioides (5 isolates) and 4 isolates as F. proliferatum and one isolate of F. pallidoroseum. Identification of the isolates was made on the basis of morphological characters and as per identification report received from ITCC (Indian Type Culture Collection) (Table-1), IARI New Delhi. The report reveals three different species of Fusarium viz. F. verticillioides, F. proliferatum and F. pallidoroseum. Out of three species of Fusarium, two species viz. F. verticillioides and F. pallidoroseum have already been reported while F. proliferatum is new record and addition in the list of stalk rot complex pathogens of maize. The isolates of Fusarium spp . collected from different locations showed variations, in basic culture characterist ics. However, differences are minor but it shows the variabilit y wit h t he geographical area and climate. The diameter and characteristics of the mycelial growth were recorded after 24, 48, and 72 hrs to know the growth pattern of all the isolates for comparison (Table 2). All the ten isolat es differed in cult ural charact ers i.e. cottony white, purple, red at bottom with red cottony, white grey, purple violet, cottony pink, cottony white with violet ring at center, cottony white with bluish at center, dirty white cottony and dirty grey color growth were observed in Fv -01 to Fv-10 respectively. Colony diameter of isolates ranged between 54.70 to 90 mm. The mycelial growth in isolate Fv-01 completed earlier hence, observations were recorded to know the relative differences in radial growth. The average radial growth of isolate Fv-01 ( F. verticillioides ) was highest i.e. 90.00 m m followed by Fv-02 (F. verticillioides) 86.44, Fv-06 (F. Proliferatum) 84.65, Fv-08 ( F. verticillioides ) 80.32, and Fv-07 (F. proliferatum) 76.40, while, in isolat e Fv -03 ( F. p allid oroseu m ), Fv -05 ( F . proliferatum), Fv-04 ( F. verticillioides), Fv-10 (F. verticillioides) and Fv-09 (F. proliferatum) it was comparatively less i.e. 72.28, 71.50, 62.80, 60.22 and 54.70 mm respec tively at 7th day of incubation under uniform environments and media. Isolates varied among themselves, with respect to pigmentation viz white 3 (number of isolates), pink 2, violet 2, pale yellow, light brown and brown 1. Margin and topography of the isolates varied from circular 6, semi circular 3, waivy 1 and flat 5, medium raised 3 and raised 2, 116

M K Khokhar et al. Table 3. Variation in conidial morphology, measurement, septation and spore count of Fusarium spp. isolates.

Fusarium spp.

Isolate designation

Spore size (µm)* Macro conidia Micro conidia Leng Width Lengt Width th h

Septation in macro

No. of spore/ml*

conid-

Macro conidia

Micro conidia

Fv-01


40.24

6.60

10.33

1.50

5-7

3.4x105

3.8x105

Fv-02


37.42

4.89

9.82

1.80

4-7

2.9 x105

3.1 x105

Fv-03 Fv-04

Fusarium pallidoroseum Fusarium verticillioides

29.50 21.04

2.28 4.14

4.20 5.62

1.83 2.44

3- 5 4-5

2.6 x105 2.0x105

2.8 x105 2.7 x105

Fv-05


22.55

3.89

10.10

4.05

3-5

2.2 x105

3.0x105

Fv-06 Fv-07

Fusarium proliferatum Fusarium proliferatum

18.83 17.66

2.06 3.08

6.64 4.56

1.60 2.96

3-4 3-4

2.5 x105 1.5 x105

3.2 x105 2.5 x105

Fv-08


38.14

5.04

12.43

3.09

4-6

2.2 x105

3.0 x105

Fv-09 Fv-10

Fusarium proliferatum Fusarium verticillioides

17.57 10.92

1.80 2.78

9.09 6.17

1.48 2.07

3 -4 2-3

0.8 x105 2.2 x105

1.6 x105 3.0 x105

SEm+

0.10

0.016

0.03

0.01

0.008

0.010

CD at 5%

0.28

0.045

0.08

0.03

0.024

0.029

CD(P=0.01)

0.37

0.06

0.11

0.04

0.031

0.038

*

Mean no. of 50 conidia and ± S.D. of mean value

respect iv ely . All t he t en isolates of F. verticillioides showed significant variations in conidial morphology. Results presented in Table3 show that mean length and width of macro conidia in different isolates of Fusarium spp. ranged from 10.90 – 40.42 x 1.80 – 6.60 µm and the mean length and width of micro conidia in different isolates ranged from 4.20 – 12.43 x 1.50 – 4.5µm. Macro conidia were produced by all the isolates but their size varied between 40.24 to 6.60 µm. Similarly all the isolates produced micro conidia with considerable variations i.e. 10.92 2.78 µm. Among the Fusarium isolates, the maximum length and width of macro conidia w a s r e c o r d e d i n t h e i s o l a t e F v -0 2 w h i c h measured 40.24- 6.60 µm followed by isolate Fv08 with 38.14- 5.04, Fv-01 with 37.42- 4.89 µm, while minimum in Fv -09 as 17.57- 1.80 µm. Among the Fusarium spp. isolates, the maximum length and width of micro conidia was recorded in the isolate Fv-08 which measured 12.43- 3.09 µm followed by isolate Fv-02 with 11.33- 1.50 µm, while minimum in Fv-03 with 4.20- 1.83 µm. Excellent sporulation of macro and micro conidia was found in isolate Fv-01 3.4x10 5 and 3.8x105 , followed by Fv-02 with 2.9 x105 and 3.1 x105, Fv03 as 2.6 x105 and 2.8 x105 and Fv-06 having 2.5 x105 and 3.2 x105, while minimum sporulation of macro conidia was observed in Fv-09 with 0.8 x10 5 and 1.6 x10 5 . T here was considerabl e variation with respect to number of septa in the range of 2-7 (Table 3). The conidial morphology

of Fu sa ri um v er ti ci ll ioi de s is ol at es a re i n agreement with those described by Sidddiqi et al. 1995, Hirata et al. 2001, Patil et al. 2007. Variability is one of the natural phenomenon which largely depend on climatic conditions, geography and cropping pattern. The survival of pathogen largely depends on variability as it is a continuous feature found in nature. Studies on variability in the pathogen and host ar e important for documenting resistant sources. Molecular variability (RAPD) In the present study, the genetic variation among ten isolates of F. verticillioides by PCR amplication using ten ramdom operon decamer primers showed polymorphism and representative RAPD profile is depicted in Fig 1. All these 10 primers showed polymorphism. Whereas, primer OPG-16 generated maximum 12 bands in the range of 200bp to 1780bp. A total of 80 amplified bands were obtained and 47 were polymorphic. The DNA amplification and polymorphism generated among various genotypes of Fusarium spp. using RAPD primers are presented in Table-4. The total number of bands observed for every primer was recorded separately and polymorphic band percentage was calculated subsequently. The total number of amplified bands varied between 5 (primer OPG7) and 12 (primer OPG-16) with an average of 7.5 bands per primer. The polymorphism observed as high as 87.5% for OPA-16 and OPC-4 to as low as 28.6% for OPC-6, these results exhibit the 117

Morphological and Molecular Characterization of Fusarium Table 4. DNA amplification profile and polymorphism generated in different isolates of Fusarium spp. using

Primer

OPA-6 OPA-9 OPA-16 OPA-19 OPC-2 OPC-4 OPC-6 OPG-7 OPG-16 OPG-17 10 Total

Sequence (5`→3`)

TGCCGAGCTG AATCGGGCTG AGCCAGCGAA TGGGGGACTC TCCGCTCTGG AGGGAACGAG ACCCCCGAAG GTGAGGCGTC CCGCATCTAC ACGACCGACA Total Avg.

Total No.of Bands 8 6 8 7 8 8 7 5 12 11 80 8.0

No. of Polymor-morphic Bands 3 4 7 5 3 7 2 3 5 8 47 4.7

importance of RAPD primers for diversity analysis. Average polymorphism across all the 10 isolates was found to be 59.1% and overall size of PCR amplified products ranged between 200bp to 2275bp. The PIC values provide an estimate of the discriminatory power of a locus or loci by taking into account not only the number of polymorphic bands that are expressed, but also the relative frequencies of these polymorphic bands. The PIC values ranged from as low as 0.12 in primer OPG-7 to as high as 0.35 in primer OPC-4 with an average of 0.20 for all ten primers. In present investigation primer OPC-4 was most informative followed by OPG-17 and OPA-19 whereas, primer OPA-6 and OPA-16 were least informative. Similarity matrices based on RAPD data Similarity coefficient among 10 isolates of Fusarium spp. based on DNA amplification using RAPD markers was estimated using Jaccard coefficient of similarity (Table 5) (Sneath and Sokal 1973) and thus similarity matrix was generated. The similarity coefficient ranged from 0.105

Number of Monomorphic Bands

Polymorphisms (%)

5 2 1 2 5 1 5 2 7 3 33 3.3

37.5 66.7 87.5 71.4 37.5 87.5 28.6 60.0 41.7 72.7 59.1 Avg.

PIC Values

0.12 0.24 0.15 0.26 0.15 0.35 0.045 0.25 0.18 0.32 0.20 Avg.

to 0.848 i.e. 74.08 –84.80% or the genetic diversity ranged from 10.5- 100%. The average similarity across all the genotypes was found to be 0.371 showing that the genotypes were highly diverse genetically. Maximum similarity value of 0.848 was observed between isolate F-5 and F-4. Isolate F-1 and F-6 was found to be genetically diverse with minimum similarity value of 0.105. The dendrogram explains diverse group of Fusarium spp. which is also clear from identification report. Hence, variability exists in the maize growing areas with narrow distant locations, which may affect the durability of any resistant variety/ varieties against PFSR. Dendrogram was constructed based on molecular data generated by 10 RAPD primers using NTSYS Pc (numerical taxonomic and multivariate analysis system) version 2.02 e (Rohlf, 1998) UPGMA clusters analysis grouped Fusarium spp. isolates into two clusters with 0.28% similarity coefficient. The first minor cluster comprised only one Fusarium spp. (Fv-01). While, the major cluster subdivided into two sub cluster at 0.44% similarity coefficient. The sub

Table 5. Jaccard’s similarity matrices coefficient produced using 10 RAPD Primers with UPGMA analysis

118

M K Khokhar et al.

cluster I comprised four Fusarium spp. viz. Fv-02, Fv-03, Fv-04 and Fv-05. Further, sub cluster II subdivided into two minor clusters at 0.62% similarity coefficient with remaining Fusarium species. These results exhibit the importance of RAPD primers for diversity analysis. It is difficult to distinguish the different isolates of Fusarium using traditional morphological basis to identify. To understand the existence of genetic variation among the isolates of Fusarium spp., PCR based technique i.e. RAPD was used in the present investigation. RAPD studies also help to know the specific markers for the identification of particular race or detection of pathogen (Sabir 2006). The suitability of random amplified polymorphic DNA to detect the variations among the isolates of Fusarium spp. was evaluated. In the present investigation, OPA, OPC and OPG series primers were used to determine genetic difference between isolates and to construct dendrogram (Fig 1). The total number of amplified bands (A total of 80 amplified bands were obtained and 47 were polymorphic) varied between 5 (primer OPG-7) to 12 (primer OPG-16) with an average of 8.0 bands per primer. Of the 10 primers used for amplification OPC4 and OPA16 showed 85.5 per cent polymorphism. Information on banding pattern for all the primers was used to determine genetic difference between isolates and to construct a dendrogram. Similarity coefficient ranged from 0.28 to 0.95 per cent. The dendrogram by RAPD data revealed that the ten isolates were broadly differentiated into two clusters I and II. Cluster I comprised only one Fusarium spp. (Fv-01) and cluster II further was sub grouped in to sub clusters i.e. I and II comprising remaining species (Fig 2). In a similarly study, Assigbetse et al. (1994) observed genetic diversity within a collection of 46 isolates of worldwide origin, based on pathogenic and RAPD markers and established DNA fingerprinting for race characterization. Sivaramakrishnan et al. (2002) studied genetic variability in 36 isolates of Fusarium udum collected from 4 pegionpea growing states in India and analyzed by using RAPD and AFLP technique. They suggested that PCR based method to identify the different races of Fusarium wilt pathogen will serve the purpose of routine analysis of field population and drawing a pathogen map of the country. Similarly, Gherbawy et al. (2002) used RAPD technique for the identification of F. subglutinans, F. proliferatum and F. verticillioides isolates from maize in Australia. The isolates from different locations which were studied for morphological variability could be differentiated further by molecular variability studies. In the present study, 10 primers screened

for amplification of DNA of various isolates of Fusarium species and all the primers were found to give reproducible bands with high percentage of polymorphism. Diverse gene pool in fungal genera Fusarium with high genetic variability existed among the recovered species as revealed by RAPD markers. Understanding the genetic structure of pathogen populations in present study may provide insights into the epidemiology and evolutionary potential of pathogen and could lead to improved strategies for controlling the disease. The results of the present investigation revealed that, isolates from same geographical locations were closely related and the isolates from different locations are genetically more diverse. Similar work was carried out by Haung et al. 1997, Kini et al. 2002, Abd El-Slam et al. 2003, El-Fadly et al. 2008, Ono et al. 2010. This signifies that F. verticillioides isolates are widely dispersed across the state which could be possibly attributed to the high sporulating nature of the pathogen, its mode of dispersal and the free movement of seed throughout the state. Besides their difference at species level in Fusarium, it has been found that these isolates may have developed variation due to climate change, cultivar, soil characters as well as cropping pattern followed. In conclusion, the present study provides information about the causal agents of post flowering stalk rot of maize in India and reveals for the first time genetic and molecular variability among Fusarium isolates belonging to three Fusarium species (F. proliferatum, F. pallidoroseum and F. verticillioides) obtained from different geographic regions in Rajasthan (India). In addition the present study generated significant information in terms of cultural and morphological variability of Fusarium verticillioides which could be further used by breeders for evolving region specific resistant varieties of maize. References Abd El-Salam KA Schneieder F Khalil M S Aly AAand Verreet JA (2003). Genetic variation at the Intra- and Inter specific level in Fusarium spp. Associated with Egyptian cotton. ZPjlkrankh Pflschutz 110: 46-53. AICRP (2013). Annual Report of AICRP Maize Pathology Udaipur center. Pp-27. Assigbetse KB Fernandez MP Dubois D and Geiger JP (1994). Differentiation of Fusarium oxysporum f. sp. vasinfectum races on cotton by random amplified polymorphic DNA (RAPD) analysis. Phytopathol 84:622-626.

119

Morphological and Molecular Characterization of Fusarium

Booth C (1971). The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. 237. Dorn B Forrer HR Schurch S and Vogelgsang S (2009). Fusarium species complex on maize in Switzerland: occurrence, prevalence, impact and mycotoxin in commercial hybrids under natural infection. Eur J Plant Path 125:51-61. Doyle JJ and Doyle JL (1990). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19:11-15. El-Fadly GB EI-Kapaz MK Hassan MAA and El-Kot GAN (2008). Identification of some Fusarium spp. using RAPD-PCR Technique. Egypt J Phytopath 36: 71-80. Gherbawy YA MH Alder A and Prillinger H (2002). Genotypic identification of F. subgluttnans, F. proliferatum and F. verticillioides strains isolated from maize in Australia. Mycologia 30:139-45.

194. Leslie JF and Summerell BA (2006). The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, USA. 388. Nur Ain Izzati MZ Azmi AR Siti Nordahliawate MS and Norazlina J (2011). Contribution to knowledge of diversity of Fusarium associated with maize in Malaysia. Plant Protect Sci 47: 2024. Ono EYS Fungaro MHP Sofia S H Miguel T Sugiura Y and Hirooka EY (2010). Fusarium verticilloides strains isolated from corn feed: characterization by fumonisin production and RAPD fingerprinting. Brazilian Archives Biol Technol 53: 953-60. Pathak VN (1984). Laboratory manual of Plant Pathology. Second edition. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi: 11–12.

Haung R Galperin M Levy Y and Perl-Treves R (1997). Genetic diversity of Fusarium moniliforme detected by vegetative compatibility groups and random amplified polymorphic DNA markers. Plant Pathology 46: 871–881.

Patil AS Singh H Sharma SR and Rao GP (2007). Morphology and pathogenicity of isolates of Fusarium moniliforme causing Pokkah Boeng disease of sugarcane in Maharashtra. In: Ram R C and Sinha A .eds. Microbial Diversity: Modern Trends. Daya Publishing House, New Delhi. 234-63.

Hirata T Kimishirmj O'Donnell K (2001). lar characterization from rotten banana science 42:155-66.

E Aoki T Nirenberg HI and Morphological and molecuof Fusarium verticillioides imported into Japan. Myco-

Rahjoo V Zad J Javan NM Gohari AM Okhovvat SM Bihamla MR Razzaehian J and Klemsdal SS (2008). Morphological and molecular identification of Fusarium isolated from maize ears in Iran. J Pl Path 90: 463-68.

Iglesias J Presello DA Botta1 G Lori GA Fauguel CM (2010) Aggressiveness of Fusarium section Liseola isolates causing maize ear rot in Argentina. Journal of Plant Path 92 (1): 205-211.

Rohlf FJ (1998). NTSYS.pc. Numerical taxonomy and multivariate analysis system. (Version 2.0). Appl. Biostatistics Inc., New York.

Kaur H and Mohan C (2012). Status of post flowering stalk rots of maize and associated fungi in Punjab. Plant Disease Res 27 (2): 165-170. Khokhar MK Sharma SS Gupta R (2014). Effect of plant age and water stress on the incidence of post flowering stalk-rot of maize caused by Fusarium verticillioides. Indian Phytopath 67 (2) 143-146 Kini KR Leth V and Mathur SB (2002). Genetic variation in Fusarium moniliforme solvated from seeds of different host species from Burkina Faso based on random amplified polymorphic DNA analysis. J Phytopathol 150: 209-12. Kumar S and Shekhar M (2005). Stress on Maize in Tropics Eds. Published by Directorate of Maize Research, Cummings Laboratory, Pusa Campus, New Delhi. Angkor Publisher (P) Ltd. Noida. 172-

Sabir JSM (2006). Genotypic identification for some Fusarium sambucinum strains isolated from wheat in upper Egypt. World J Agric Sci 2:610. Saharan MS and Naef A (2008). Detection of genetic variation among Indian wheat head scab pathogens (Fusarium spp./ isolates) with microsatellite markers. Crop Prot 27: 1148-1154. Sankar NR Devamma MN Kumar VK and Giridha D (2011). First Report of Fusarium moniliforme causing fruit rot in tinda (Praecitrullus fistulosus) India. New Dis Reports 24: 24. Sharma TR Prachi and Singh BM (1999). Applications of polymerase chain reaction in phytopathogenic microbes. Indian J Microbiol 39: 79-91. Siddiqui SA Mirza JH and Haq M (1995). Studies on the growth and sporulation of Fusarium mo120

M K Khokhar et al.

niliforme Sheld., the causal organism of bakanae disease of rice: effect of nitrogen sources. Pak J Phytopath 7: 145-47.

Smith E McLaren M (1997). Effect of water stress on colonization of maize roots by root infecting fungi. African Plant Prot 3:47–51.

Sivaramkrishnan S Sethkannan SD and Singh P (2002). Detection of genetic variability in Fusarium udum using DNA marker. Indian Phytopthol 55:258-263.

Sneath PHA and Sokal RR (1973). Numerical Taxonomy. Sneath PHA and Sokal RR. (ed) San Francisco, USA, Freeman and Company.

121