“Cultural” and “morphological” variability of various cultures
Different isolates of Fusarium was collected and identified visually from the infected maize plants after survey. Based on initially growth of cultures and morphological characteristics cultures all culture sent to ITCC for identification, all isolates were identified as F. verticillioides, 2 isolates as F. proliferatum and one isolate of F. pallidoroseum, one isolate of Fusarium luffae and one isolate of Fusarium oxysporum as per identification report received from ITCC (Indian Type Culture Collection), IARI New Delhi. Out of Five species of Fusarium, three species viz. F. verticillioides, F. proliferatum and F. pallidoroseum have already been reported while Fusarium luffae and Fusarium oxysporum is new record and addition in the list of stalk rot complex pathogens of maize from Udaipur and Jaipur region. Similarly, several species of Fusarium have been identified all over the world to cause stalk rots like, F. subglutinans (F. semitectum), F. avenaceum, F. sulphureum, F. acuminatum, F. pallidoroseum, F. merismoides, F. nivale and F. solani 33, 19, 17, 39. In India, F. verticilloides, F. moniliforme, F. oxysporum, F. pallidoroseum and F. semitectum are reported to be widespread in Western Uttar Pradesh, Punjab and Rajasthan 21, 42, 43, 44,15.
Colony and morphological Characteristics
However, variation may be minor but it shows the differences with the geographical distribution, origin and area and climatic conditions of Pathogen. The measurement and characteristics of the mycelial growth of pathogen were observed after 72 hrs to record the growth pattern for comparison of all the isolates and data are presented in Table-1. All the ten isolates differentiated in vitro in cultural characters i.e. cottony colours like white, white later turned pinkish white, red at bottom with white cottony, purplish, purple violet, white grey cottony white, cottony white with violet at center, cottony white superficial, white cottony and yellowish white color growth were observed in Fv -01 to Fv-10, respectively. Colony measurements of isolates recorded between 65.80 to 90 mm diameter. Mycelial growths in isolate Fv-01 fully grown to observations were recorded to know the relative differences between radial growths of other isolates (Fig. 1). Radial growth was fast growing, in isolate JPR Fv-01 (F. verticillioides ) and UDP Fp-05 (F. pallidoroseum) were highest i.e. 90.00 mm followed by UDP Fv-07 (F. verticilloides) 89.40 mm diameter. Isolates varied in terms of pigmentation and other parameters among themselves, with respect to pigmentation viz Dirty white in JPR Fv- 01 and UDP Fv -09, white in UDP Fl- 03, pink in UDP Fv- 04, UDP Fv- 07 and UDP Fv − 06, violet in UDP Fp- 05 and UDP Fp-08, light brown in UDP Fo- 02 and UDP Fp- 10 (Fig. 1). Margin and topography of the collected isolates which varied from circular in 6, semi circular in 3, waivy growth pattern in 1 isolate and superficial growth pattern in 5 isolates, fluffy growth in 3 and raised growth in 2 isolates. All the isolates observed that significant variations were found between the morphology of Fusarium spp and data were represented in table-2. Results shows that mean length and width of macro conidia in different isolates of Fusarium spp. ranged from 15.92 to 38.30 x 1.90 to 5.90 µm or the mean length and width of micro conidia in different isolates ranged from 3.90 to 11.93 x 1.08 to 3.85µm. The highest length and width of macro conidia measured in the isolates JPR Fv -01 Which recorded 38.30 x 5.90 µm in diameter, while minimum in UDP Fp-10 as 15.92 x 2.08 µm. The maximum length and width of micro conidia was recorded in the isolate JPR Fv-01 which measured 11.93 x 2.79 µm, while minimum in UDP Fl-03 with 3.90 x 1.53µm. There was considerable differences with respect to number of present septa in the range of 2 to 6. Excellent sporulation of macro and micro conidia per ml was found in isolate JPR Fv-01 that is 3.3 x 105 and 3.9 x 105 per ml, followed by UDP Fo- 02 with 2.8 x105 per ml and 3.2 x105 per ml, while minimum sporulation of macro conidia and micro conidia were observed in UDP Fv-09 with 0.8 x105 per ml and 1.5 x105 per ml followed by UDP Fv – 04 with 2.1 x 105 and 2.6 x 105 per ml. Likewise conidial morphology of Fusarium spp. isolates were having nearly similar results where he found micro conidia were oval to club shaped with a flattened base and measured 3.32–12.43 x 1.03–4.94 µm where the macroconidia observed were typically 4–6 celled with 3–5 septa and measured 24.73–35.94 x 4.61–7.22 µm 36, 38.
At the same time, result supported by previous workers made frequent isolations of F. subglutinans and F. temperatum from infected maize stalks in maize fields in Gangwon province in 2014–2015 39. They observed mycelial growth of F. subglutinans and Fusarium temperatum 44.3 ± 0.4 and 47.60 ± 0.60 mm diameter, sequentially, on PDA medium at 25°C temperature after 4 days. Pigmentation of Fusarium subglutinans and Fusarium temperatum was alike, at first, white and orange but turning pinkish white or yellowish violet after some days. Measurement of conidial morphology, showing that Fusarium subglutinans and Fusarium temperatum engender 1×106±75.2 and 7×106 ± 34.40 spore per ml, respectively. Microconidia of F. subglutinans and F. temperatum were oval shaped, 8–20.1 and 5.4–20.9 µm long and 0–1-septate, respectively. The macroconidia of F. subglutinans and F. temperatum were 33.20–70.80 and 30.70–57.60 µm in length, respectively. Similarly, reported colony diameter in different isolates of Fusarium moniliforme or verticilloides which differ in range with in 85 to 90 mm diameter after 7 days of subculture 31.
Molecular variability (RAPD)
In the present study, the genetic variation among ten isolates of F. verticillioides by PCR amplication in through 10 operon decamer primers manifests polymorphism and illustrative RAPD profile.
Molecular variability of the ten isolates was carried out by choosing 10 RAPD markers of Operon series having 10 nucleotides in each marker. Ten primers produced scorable bands with high percentage of the polymorphism. A total of 102 bands were produced in which 78 polymorphic bands showed polymorphism of 91.6% and all the data has been represented in table-3. Among OPA series marker, OPA-11 gave 76.9% per cent polymorphism whereas OPB − 10 gave 70% polymorphism. Band size for the OPA series ranged from 0.20 kb-1kb and OPC series primer, OPC − 19 showed 80 per cent polymorphism. Among OPD series markers, OPD-01 showed highest polymorphism percent that was 91.6%. Band size had a range of 0.15-1kb for these series primers. In two of the OPF series markers, OPF-03 primer showed minimum polymorphism percent that was 62.5%.
Similarity matrices coefficient on RAPD data
Similarity coefficient among these 10 collected isolates of Fusarium spp. from different localities, based on DNA amplification using RAPD markers was estimated using Jaccard coefficient of similarity 34 and thus similarity matrix was generated. The similarity coefficient ranged from 0.123 to 0.854 i.e. genetic similarity percentage ranged between 12.3–85.40% (Table 4). Maximum similarity value of 0.854 (85.4%) was observed between isolate F-4 and F-9 followed by 0.852 (85.2%) in F-3 and F-9. Isolate F-6 and F-8 was found to be genetically diverse with minimum similarity value of 0.123 (12.3%) followed by 0.153 (15.3%) in F-2 and F-6. Dendrogram (Fig. 2) was constructed based on molecular data generated by 10 RAPD primers using NTSYS Pc (numerical taxonomic and multivariate analysis system) version 2.02 e 32, UPGMA clusters analysis grouped Fusarium spp. isolates into two clusters with 0.32% similarity coefficient. The first minor cluster comprised only two Fusarium spp. (Fv-05 and Fv-6). While, the major cluster subdivided into two sub cluster at 0.32% similarity coefficient. The sub cluster I comprised four Fusarium spp. viz. Fv-5 and Fv-06. Further, sub cluster II subdivided into two minor clusters at 0.64% similarity coefficient with remaining Fusarium species. These results exhibit the importance of RAPD primers for diversity analysis. In a similarly study, used arbitrary primers and examined 38 arbitrary primers screened for polymorphisms between the two paternal genotypes, GM1021 as resistant and GM1002 as susceptible genotypes, 23 RAPD primers showing 60.1 per cent, provided polymorphic bands suitable to differentiate between two parents28. Out of these 23 RAPD primers, OPA02 (5'- TGCCGAGCTG − 3') and pr 11(5'- CAATCGCCGT − 3') primers produced a single and strong polymorphic band at 350 and 750bp, respectively. Similarly, assessed genetic variation among ten isolates of Fusarium verticillioides by PCR amplication using ten ramdom operon decamer primers that showed polymorphism and representative RAPD profile 14. 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 found polymorphic. 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 was as high as 87.5% for OPA-16 and OPC-4 to as low as 28.6% for OPC-6; these results exhibit the importance of RAPD primers for diversity analysis. Likewise, observed genetic diversity in the Punjab populations of Fusarium moniliforme using twenty random amplified polymorphic DNA markers, in which 18 primers revealed amplification with a total number of 220 amplified fragments of DNA 12,13. There are, Primer OPT-12 showed highly polymorphic with 0.913 PIC value while OPT-9 was least polymorphic among them. The size of amplified DNA fragments ranged from 0.1-2.0 kb. Dendrogram, accused by 18 RAPD primers based on molecular study data showed that six clusters were generated with similarity percentage ranging from 36 to 96% and one independent lineage with 26% similarity coefficient values. No correlation was obtained between the genetic diversity and pathogenic variation; however the later was influenced by agro-climatic zones of north-western India which shows the effect of agro climatic conditions on the polymorphism and morphology of pathogen. This was the first attempt to study the genetic diversity of F. moniliforme causing Fusarium stalk rot of maize in Punjab. In a similarly study, 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 3. Similar work was carried out by Sivarama, they studied genetic variability in 36 isolates of Fusarium udum collected from 4 pigeon pea growing states in India and analyzed by using RAPD and AFLP technique 35. 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, used RAPD method for identify molecular variation between Fusarium subglutinans, Fusarium proliferatum and Fusarium verticillioides isolated from maize in Australia 9. Similarly observed 85 per cent polymorphism among 14 Fusarium oxysporum f. sp. ciceri isolates by using 24 RAPD primers 6. Similarily, genetic variability within 24 isolates representing seven races of Fusarium oxysporum f. sp. ciceri by RAPD, with a set of 40 primers 10. UPGMA cluster analysis cleft the different isolates into seven distinct clusters with 0.55 genetic similarities. At the same trend reported amount of genetic variation of 30 Fusarium oxysporum f. sp. ciceri isolates was evaluated by PCR amplification with a set of 40 RAPD primers 41. The result indicated that there was little genetic variability among the isolates.
Table 1
Variation in cultural characters of PFSR isolates collected from different locations
S. NO. | Isolate designation | Fusarium spp. | Colony diameter (mm) | Colony color | Margin | Topography of the culture | Pigmentation in culture | I. D. No. |
1 | JPR Fv- 01 | Fusarium verticilliodes | 90.00 | Cottony white, later turned light yellowish | Semi circular (irregular) | Superficial growth | Dirty White | 9246.13 |
2 | UDP-Fo-02 | Fusarium oxysporum | 66.44 | white, later turned Pinkish white | circular | Fluffy | Light brown | 11577.21 |
3 | UDP Fl- 03 | Fusarium luffae | 75.28 | red at bottom white cottony growth | circular | Fluffy | White | 11579.21 |
4 | UDP Fv- 04 | Fusarium verticilliodes | 65.80 | White later turned purplish | Semi circular | Slightly fluffy | Dark pinkish | 11580.21 |
5 | UDP Fp- 05 | Fusarium pallidoroseum | 90.00 | Purple violet | circular | Fluffy | Violet | 9245.13 |
6 | UDP Fv- 06 | Fusarium verticilliodes | 84.60 | White grey | | Superficial growth | Pink | 9249.13 |
7 | UDP Fv- 07 | Fusarium verticilliodes | 89.40 | Cottony white with violet at bottom | circular | Fluffy | No undulation | 9250.13 |
8 | UDP Fp- 08 | Fusarium proliferatum | 70.32 | violet (Dark purple ) | | Superficial growth | Violet | 9251.13 |
9 | UDP Fv- 09 | Fusarium verticilliodes | 75.70 | white cottony, turned light orange | circular | Fluffy | Dirty white | 9252.13 |
10 | UDP Fp- 10 | Fusarium proliferatum | 70.22 | Yellowish white | circular | Superficial growth | Brown | 9248.13 |
| CV SEM+ CD at 5% | | 3.22 1.448 4.301 | | | | | |
* Mean no. of 50 conidia and ± S.D. of mean value |
Table 2
Variation in conidial morphology, measurement, septation and spore count of Fusarium spp. isolates.
S. NO. | Isolate designtaion | Fusarium spp. | Spore size (µm)* | Septation in macro conidia | No. of spore/ml* |
Macro conidia | Micro conidia |
Length | Width | Length | Width | Macro conidia | Micro conidia |
1 | JPR Fv- 01 | Fusarium verticilliodes | 38.30 | 5.90 | 11.93 | 2.79 | 4–5 | 3.3× 105 | 3.9× 105 |
2 | UDP-Fo-02 | Fusarium oxysporum | 35.04 | 4.39 | 9.33 | 1.60 | 3–4 | 2.8× 105 | 3.2× 105 |
3 | UDP Fl- 03 | Fusarium luffae | 22.01 | 4.15 | 3.90 | 1.53 | 3–4 | 2.5× 105 | 2.7× 105 |
4 | UDP Fv- 04 | Fusarium verticilliodes | 30.00 | 2.38 | 5.52 | 2.54 | 4–6 | 2.1× 105 | 2.6× 105 |
5 | UDP Fp- 05 | Fusarium pallidoroseum | 21.50 | 3.99 | 9.90 | 3.85 | 3–5 | 2.1× 105 | 3.2× 105 |
6 | UDP Fv- 06 | Fusarium verticilliodes | 17.80 | 2.03 | 5.94 | 1.30 | 3–5 | 2.4× 105 | 3.3× 105 |
7 | UDP Fv- 07 | Fusarium verticilliodes | 17.60 | 3.07 | 3.96 | 2.66 | 3–5 | 1.6× 105 | 2.5× 105 |
8 | UDP Fp- 08 | Fusarium proliferatum | 37.13 | 4.14 | 10.12 | 1.50 | 4–5 | 2.1× 105 | 3.2× 105 |
9 | UDP Fv- 09 | Fusarium verticilliodes | 16.07 | 1.90 | 8.99 | 2.09 | 3–5 | 0.8× 105 | 1.5× 105 |
10 | UDP Fp- 10 | Fusarium proliferatum | 15.92 | 2.08 | 6.13 | 1.08 | 2–4 | 2.3× 105 | 3.1× 105 |
SEm± | 0.125 | 0.013 | 0.028 | 0.008 | | 0.021 | 0.025 |
CD at 5% | 0.347 | 0.037 | 0.079 | 0.023 | | 0.008 | 0.009 |
CV | 3.52 | 2.77 | 2.66 | 2.80 | | 2.43 | 2.21 |
Table 3
Similarity coefficient values in RAPD for PFSR isolates
| PFSR − 2 | PFSR-3 | PFSR-4 | PFSR-5 | PFSR-6 | PFSR- 7 | PFSR − 8 | PFSR − 9 | PFSR − 10 |
PFSR − 2 | 1.000 | | | | | | | | |
PFSR − 3 | 0.820 | 1.000 | | | | | | | |
PFSR − 4 | 0.652 | 0.633 | 1.000 | | | | | | |
PFSR − 5 | 0.253 | 0.252 | 0.321 | 1.000 | | | | | |
PFSR − 6 | 0.153 | 0.451 | 0.250 | 0.327 | 1.000 | | | | |
PFSR − 7 | 0.222 | 0.444 | 0.310 | 0.754 | 0.412 | 1.000 | | | |
PFSR − 8 | 0.253 | 0.532 | 0.421 | 0.254 | 0.123 | 0.624 | 1.000 | | |
PFSR − 9 | 0.320 | 0.852 | 0.854 | 0.432 | 0.222 | 0.325 | 0.352 | 1.000 | |
PFSR-10 | 0.425 | 0.352 | 0.652 | 0.251 | 0.602 | 0.425 | 0.124 | 0.752 | 1.000 |
Jaccard’s similarity matrices coefficient prodused using 10 RAPD Primers with UPGMA analysis |
Table 4
RAPD banding profile of different primers for different isolates of PFSR
S. No. | Primer | 5’-3’ Sequence | RAPD scored Bands | RAPD Polymorphic scored bands | Polymorphism (%) |
1 | OPA11 | CAATCGCCGT | 13 | 10 | 76.9 |
2 | OPB10 | CTGCTGGGAC | 10 | 7 | 70 |
3 | OPC19 | GTTGCCAGCC | 10 | 8 | 80 |
4 | OPD01 | ACCGCGAAGG | 12 | 11 | 91.6 |
5 | OPD04 | TCTGGTGAGG | 9 | 6 | 66.6 |
6 | OPD07 | TTGGCACGGG | 13 | 11 | 84.6 |
7 | OPF03 | CCTGATCACC | 8 | 5 | 62.5 |
8 | OPF14 | TGCTGCAGGT | 7 | 6 | 85.7 |
9 | OPG09 | CTGACGTCAC | 11 | 7 | 63.6 |
10 | OPG16 | AGCGTCCTCC | 9 | 7 | 77.7 |
Total | | | 102 | 78 | |
Per cent Polymorphism = No. of polymorphic bands / Total number of bands *100 |