Survey and variability among Fusarium oxysporum f. sp. lini isolates inducing wilt disease of linseed in Vidarbha growing regions of Maharashtra

Introduction

Linseed is one of the oldest crop cultivated for its seeds and fiber. The two products of seed are linseed oil and linseed meal. Flax (Linum usitatissimum) is also one of the richest dietary sources of α-linolenic acid (ALA) and is a good source of soluble fiber mucilage in human nutrition. Linseed is very rich in oil and the oil and protein percent in seed of linseed varies from 37.8 to 43.2% and 20.00 to 24.8% respectively. During 2023-24, a total of 1.67 lakh tonnes of linseed was produced in India from an area of 2.39 lakh ha with an average productivity of 698 kg/ha. In terms of productivity, Rajasthan stands first (1072 kg/ha) followed by Bihar (851 kg/ha) and Nagaland (771 kg/ha) (Linseed, Annual report 2023-24). Among the fungal diseases of linseed, Alternaria blight, Powdery Mildew, Rust, Wilt is a major constraint responsible for low production and productivity along with rust and powdery mildew (Kishore et al. 2011). The crop loss due to linseed wilt incited by Fusarium oxysporum f. sp. lini is the most destructive disease (Sattar and Hafiz (1952) reported losses by wilt disease in linseed crop up to 80 % under favourable conditions. Linseed wilt incited by Fusarium oxysporum f.sp. lini (Bolly) Snyder and Hansen was first reported by Luggar (1890) from Minnesota, USA. In India, disease was first reported by Pearl (1923) in Madhya Pardesh. Flax wilt is caused by the soil-and seed-borne fungus Fusarium oxysporum f. sp.lini (Bolley) Snyd. and Hans. This pathogen invades the roots, mainly through root hairs, develops in xylem vessels and becomes systemic. Disease symptoms may be obvious during the early seedling stage or may appear later. The pathogen infects the plant through the roots and colonizes the vascular system, leading to wilting, stunting, and ultimately, plant death. The disease spreads through soilborne inoculum, including infected plant debris and soil particles. The mycelium is septate, branched, and intracellular. The pathogen produces branched, hyaline, and short conidiophores that give rise to hyaline and micro and macro conidia. The conidia are both micro-and macroconidia. Favorable environmental conditions, such as high soil moisture and warm temperatures, promote disease development and progression. The pathogen with its high saprophytic ability can survive in soil for a long period during which it may have to go through different environmental stresses and biological competition which may lead to the existence of physiologic races. The most effective and practical method of control worldwide is to use fungicides (Gupta et. al., 1988) [18] or resistant cultivars. However, the effectiveness of host resistances is curtailed by the occurrence of pathogenic races in F. oxysporium f.sp. lini (Jimenez-Gasco et.al., 2004) [8] . Researchers from all across the world suggested creating F. oxysporum as a global model for the knowledge of fungal virulence. The various isolates of Fusarium have been classed on the basis of their nutritional demands, temperature, sensitivity to chemicals (Khare et al 1980) [9] and morphology and aggressiveness. Increased attention is being paid by the scientists to develop wilt resistant varieties for large scale cultivation of Linseed and appropriate management of the disease. Therefore, accurate and rapid identification of pathogen is necessary. Identification of pathogenic races would be done by use of different reaction to selected host genotypes. To reduce the effects caused by the variability in the pathogen it is essential to know the genetic nature and pathogenic types of isolates prevailing in linseed growing areas of India. Hence, the present investigation will help to understand the variability in the pathogen which will be useful for its exploitation in wilt resistance breeding and further used for development of area specific resistant varieties of linseed. The present study was conducted to correlate cultural and morphological variability in F.oxysporum isolates collected from major linseed growing areas of India. The study will help to know the prevalence and distribution of more virulent isolates of wilt pathogens in the country. The identified virulent isolates will be used for screening of breeding material of linseed and the obtained resistant lines will be supplied to linseed breeders to develop wilt resistant linseed varieties with high yielding potential. The introduction of wilt resistant linseed cultivars will help to reduce losses caused by wilt disease and thus enhance the linseed production in the country.

Materials and Methods

Survey and collection of samples

During two year Rabi cropping season, 2021-2022 and 2022-23 at 8 districts viz., Akola, Nagpur, Wardha, Bhandhara, Gondia, Amaravati, Gadchiroli, Chandrapur surveyed for the assessment of disease incidence. Linseed plants naturally infected and showing typical wilt symptoms were collected from farmer's fields from different locations in different district of Maharashtra and brought to the laboratory. At each field, three observations on total number of linseed plants in l m 2 area and total wilted plants in the sampling area were recorded to calculate per cent disease incidence of wilt in the field by using formula (Mayee and Datar, 1986) [11] total number of plants per spot as [(number of diseased plants/total number of plants) x 100]. Samples were placed into paper bags and proper labeled to indicate location, sample number and date of collection. The samples collected during inspection were brought to Plant Pathology Section, College of Agriculture, Nagpur Isolation and purification of pathogen Isolation of Fusarium oxysporum f. sp. lini isolates collected from different locations, from wilted linseed plant roots/stem were made on nutritional artificial media (PDA). The causal organisms were isolated from root of plant that affected by wilt disease. The roots and stems of infected plants were washed in tap water to remove adhering soil particles, if any and root bark was removed before isolation to avoid contamination. The roots and stems were split open and small bits (size 2.5cm) were cut with sterilized sharp blade. These bits were then disinfected with 0.1% solution of mercuric chloride for one or two minutes, then washed thoroughly in sterile distilled water thrice to remove the traces of mercuric chloride, dried in sterile blotter paper and aseptically transferred on PDA in petriplate, and incubated at 25±2 °C for a week. Fungus growth in plate was examined and then sub-cultured on PDA slants. By frequent subculturing, it was purified and maintained on PDA slants for further studies. A total of 24 isolates of pathogen were isolated from available samples. Isolation was made and the isolation culture was purified by following single spore isolation techniques (Choi 1999). A little bit of fungal growth with spore placed was in a test tube containing 5 ml of sterilized water and shaken well to prepare 60 suspension of spore. A dilute spore suspension was poured on water agar media. Petri dishes to form a very thin layer on it and spores allowed settling down on the agar surface. Settled spores were separated out from each other, selected under the microscope. The position of single spore was marked help of plastic rod on glass surface under low power. The plates were taken to the isolation chamber and cut of agar from marked areas were cut of the help of dummy cutter in Petri dish and transferred to Petri dishes containing sterilized PDA medium. After proper growth of fungus obtained by single spore culture regular sub-culturing was done to check contamination, till pure cultures were obtained. The culture was then transferred into the incubator at 25±2 ºC temperature and growth observed after four days.

Morphological and cultural characteristics

The isolates of Fol collected from different linseed growing states of India were studied for their morphological characterization. All 24 isolates were grown on potato dextrose agar (HiMedia, Mumbai, India) medium for morphological studies comprising viz., size of macroconidia, microconidia & septation of all the isolates, 5 mm diameter disc of mycelia of each isolates were taken from the actively growing culture and placed upside down centrally on 90 mm petridish containing solidified PDA medium and the inoculated plates were incubated at 25±2 °C for 9 days. Each plate was replicated three times. The spore dimensions such as length and width of both the microconidia and macroconidia were measured through a calibrated compound microscope (Procam software) for 50 spores of each isolate and the mean was recorded as final dimension. Septation of the macroconidia was also recorded. The isolates were also cultured in liquid media in 250 ml flask containing 100 ml of potato dextrose broth (PDB). These flasks were incubated at 25±2 °C for 15 days. The colony characters, sporulation and pigmentation of all the isolates were also recorded. The incidence of linseed wilt was recorded in four district Akola, Chandrapur, Wardha, Nagpur (Linseed annual report 2023-24). The incidence of linseed was recorded in Bhandhara, Gondia, Gadchiroli, Amaravati (Linseed annual report 2022-23). Similar finding incidence of linseed wilt was recorded in three districts like Latur, Beed and Osmanabad (Ranganath, 2016).

Results and Discussion

Variability in morphological characteristics

All isolates of Fusarium oxysporum f.sp. lini differed in their morphological characteristics when grown on PDA. Morphological studies revealed variation in size of microconidia, macroconidia and number of septa among twenty-four isolates of Fusarium oxysporum f.sp. lini. The results presented in Table 02 indicate that all isolates of Fusarium oxysporum f.sp. lini used in study varied significantly in their morphological and cultural characteristics on PDA.

(A) Macro conidia Variation was observed in length of macroconidia. The average length of all twenty-four isolates varied from 6.98 µm to 32.98 µm and average breadth 1.27 µm to 8.01 µm. Minimum average size was recorded in the isolate Fol-9 (8.05 x 1.77 µm) while the maximum average size was recorded in the isolate Fol-3 (25.13 × 6.24 µm) length and breadth respectivly. Numbers of septa observe varied from between 2-7 in all isolates (Plate 3). In similar studies Saharan and Mehta (2002) [14] stated that both micro and macroconidia were produced by isolates of Fusarium oxysporum f.sp. lini. The average size of microconidia ranged from 4.8 -14.4 x 2.2 -4.8 μm and in macroconidia the average size ranged from 21.0 -53.0 x 2.4 -5.6 μm. Dubey et al. (2010) [15] observed isolates of F. oxysporum f.sp. ciceris to vary with respect to their conidia size. Microconidia varied from 5.1 -12.8 x 2.5 -5.0 μm in size, whereas macroconidia were from 16.5 -37.9 x 4.0 x 5.9 μm with 1-5 septations most commonly with 2-3 septate conidia. Similar finding were observed by other researchers to viz., Gupta et al. (2011) [7] , Kriplani et al. (2018) [10] .

(B) Micro Conidia

Variation was observed in length of microconidia. The average length of all twenty-four isolates varied from 2.20 µm to 10.34 µm and average breadth 0.97 µm to 2.35 µm. Minimum average size was recorded in the isolate Fol-22 (2.20 x 0.97 µm) while maximum average size was recorded in the isolate Fol -17 (10.34 x 2.35 µm). Number of septa in microconidia were 0 in all isolates (Plate 4). In similar studies Saharan and Mehta (2002) [14] stated that both micro and macroconidia were produced by isolates of Fusarium oxysporum f.sp. lini. The average size of microconidia ranged from 4.8 -14.4 x 2.2 -4.8 μm and in macroconidia the average size ranged from 21.0 -53.0 x 2.4 -5.6 μm. Fusarium oxysporum f.sp. ricini isolate of castor wilt (Desai et al. 2003) [4] , Fusarium oxysporum f.sp. carthami isolate of Safflower wilt (Prameela et al. 2005) [12] and Fusarium oxysporum f.sp. ciceri isolate of Gram wilt (Trivedi and Gurha, 2007).

Variability in Cultural Characteristics (A) Variability in mycelial appearance of Fusarium oxysporum f.sp. lini isolates

The Fusarium oxysporum f.sp.lini isolates selected for cultural studies showed variation on PDA medium in table 3, (Plate 5). The observation of mycelial colour were taken on 7th day after inoculation. The mycelium growth of Fusarium oxysporum f. sp. lini was observed varied from fluffy, appressed flat, partially appressed, partially flat appressed. fluffy growth was observed in isolates FOL [17] and Tiwari and Dhar (2011) [3,18,6,13] .

Variation in the radial growth rate of Fusarium oxysporum f.sp. lini isolates On the basis of radial growth, the isolates of F. oxysporum f.sp.lini were categorized into 3 groups viz., fast growing (more than 70 mm), moderate growing (50 -70 mm) and slow growing (less than 50 mm) on PDA medium( FOL 11, FOL 13, FOL 14, FOL 15, FOL 17, FOL 18, FOL 19, FOL 20, FOL 23, FOL 24) were fast growing (Fig. 1). Similar variations in colony growth were recorded earlier by Prameela et al. (2005) [12] among the isolates of Fusarium oxysporum f.sp. carthami. Trivedi and Gurha (2007) grouped the isolates of Fusarium oxysporum f.sp. ciceri on the basis of mycelium growth i.e. fast, medium and slow. Similarly, Wagh et al. (2009) also observed variability in growth among different isolates of Fusarium oxysporum f.sp. lini.

(B) Variation in pigmentation of different isolates

The isolates varied in their pigmentation on 7 th day after inoculationon PDA on reverse side of PDA plates. Light yellow to purple pigmentation was observed in isolates FOL The present results similar with the findings of Madhukeshwara and Seshadri (2001) [17] who studied the existence of variability among six isolates of F. udum with respect to morphological and cultural characteristics i.e. varied pigmentation was noticed from white to dusk red. Rangaswamy et al., (2016) [9] studied that F. udum isolates were having considerable variation in substrate pigmentation for all the isolates. study revealed high degree of variations among them macroconidia mean size range from length (8.05 -25.13 µm) and breadth (2.01 -6.24 µm). The mycelial growth on PDA was observed varied from fluffy, appressed flat, partially appressed, partially flat appressed mycelial growth with or without serrate margin and three types mycelial colour were observed white, dull white, cottony, dark white and Pigmentation light yellow to purple, light yellow, light orange to purple orange, White in different isolates of F. oxysporum f.sp. lini on PDA medium. Based on radial growth, isolates were categorized as fast growing (17 isolates), moderate growing (07 isolates).

Acknowledgement

I am thankful to my guide Dr. R. W. Ingle for constant support. And also grateful to the Head of the Plant Pathology section, College of Agriculture, Nagpur for providing all the necessary facilities for conducting the research work.

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