ISSN: 2454-1362, ... · Imperial Journal of Interdisciplinary Research (IJIR) Page 1243 ......

Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-9, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Imperial Journal of Interdisciplinary Research (IJIR) 243

Histopathological Studies of Fusarium moniliforme Causing Root Rot of

Sorghum bicolor L.

Mahesh, H. M., Sharada, M. S., Angel Catherine and Divya, C. R

Department of Studies in Botany, University of Mysore, Manasagangothri, Mysuru-06

Abstract: Histological observations of Fusarium moniliformae in sorghum in causing the Root rot of Sorghum are described. Pathogen invades into the cortex region of the living tissues of the root and Systematic invasion of a susceptible variety by a pathogen was effective and the mechanism was complete in infecting the host and spread the disease and also to complete the cycle of the pathogen. The pathogen was treated to the host at different intervals of time viz., 2, 4, 6, 8, 10, 12, and 24 hrs and the process of infection was recorded. The mode of infection described from 2 hrs, as it infects the outer epidermis and later hours thick hypahe is seen then the pathogen enters into adjacent cells at 8 hours increased mycelia growth thorough the host cells. Development of conidia and disintegration of host cells showing collapsed tissues and finally formation of papillae also with the hypersensitive reaction is seen. After 24 hrs mycelia ramification of the host tissue and complete growth of the mycelia is seen. Then the host dies finally after 24 hours of infection by Fusarium moniliformae.

Keywords : Hypersensitive Reaction, Root rot, Maceration.

Introduction

Fusarium is one of the most cosmopolitan of the plant pathogen is found in soil wherever we grow sorghum. Fusarium root rot on sorghum typically involves the cortical tissues first and then the vascular tissues of the root [33] The infected sorghum root rot if often uprooted. The use of certain staining techniques can facilitate considerable observations and experimental research on plant pathology, by allowing plant and fungal tissues to be differentiated. Sorghum (Sorghum bicolor L. Moench), a member of the grass family Graminea, is a hardy plant able to grow under a variety of field conditions, and which, together with maize, barley, wheat, rice and sugarcane, forms part of the world’s feed and food production chain for animal and human

consumption [8,15]. India contributes about 16% of the world’s sorghum production. This crop was one of the major cereal staple during 1950’s and occupied an area of more than 18 million hectares then reduce to 5.72 million hectares in 2013-2014 (TE 2014). Production increases from 9 million tons in the early 1970s to 12million tons in early 1980 and maintained this level for over a decade until early 1990s, followed by a steep decline to 10.62 million tons 2013-2014. Despite the decrease in area over the year, production has been sustained at 10.62 million tons due mainly to adoption of improve varieties and hybrids. Among the states, Maharashtra alone recorded positive growth in production during both kharif and rabi seasons, while Karnataka registered a positive growth rate in rabi production. Gujarat recorded highest growth in yield of 3.72%, while it also recorded highest decline in area in kharif. Based on the performance of sorghum in Maharashtra, it appears that relatively it has a promising future. This therefore prompts the need for the study on the need for the profitability of the enterprise so as to ascertain its viability among the producing states in two different seasons (ie 2004-05 and 2010-2011)[27]. Severe damage to the roots can result in decreased water absorption, nutrient uptake [6] and loss of anchorage through the destruction of older roots [33].This can lead to plants being easily uprooted, lower grain yield, a reduction in drought tolerance and eventually plant death [33] Lesions caused by Fusarium spp. can differ in size, from small circular spots scattered over the roots or stripes that extend over most of the root surface. These are usually red to purple in colour [6] depending on the host genotype, although light brown or black discoloration may be associated with tan plant types. The infection may cause a breakage of the lateral roots from the main root system and infected roots generally lack root hairs [17] Degeneration of the vascular bundles and pith are rarely caused by Fusarium spp. on roots, but

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may be caused by secondary or opportunistic organisms that are usually present and can cause the disintegration of tissues [6] e.g. Periconia spp. and other Fusarium spp. [23]. When disintegration of the inner tissues occurs, all that remains is a dry hollow shell that lacks any form of structural integrity [29].

The main approach for the Fusarium classification is still morphology, and the primary trait for species to be placed in Fusarium genus is the occurrence of the asexual spores, the distinctive banana-shaped macroconidia. The efficacy of composts in the suppression of root rots depends on the composition of the compost. Compost with peat-based growing medium with chitin, increases the growth of cucumber in the absence of root pathogens. In treatments where the root and stem rot pathogen, Fusarium oxysporum f. sp. radicis-cucumerinum, was present, a higher disease incidence occurred with composting compared to the absence of composts. This was attributed to the breakdown of chitin that releases ammonia which enhances susceptibility of the cucumber to root and stem rots [25]. A mono-phialide is a conidiation cell with a unique pore from which the endoconidia are released; a polyphialide can possess several such openings. Microconidia can vary in shape and size, and are produced in the aerial mycelium in clumps or chains, both on monophialides and polyphialides. Finally, chlamydospores are resistant structures with thickened walls and high lipid content, in the case of their presence, they can form in the middle of the hyphae or at their termini.

MATERIALS AND METHODS

Isolation and identification of Pathogen from the Sorghum bicolor samples.

Collection of plant material

Sorghum seeds, leaves, roots and leaves showing symptoms of root rot disease were collected from different regions of Mandya district. The sorghum seeds were collected from agricultural farm National Seed Corporation, public and private seed agencies of Mandya districts.

Screening Isolation and identification of the Pathogen Fusarium moniliformae

Rhizospheric soils of Sorghum field looking diseased irrespective of plant growth stages were collected from different Sorghum growing different regions of Mandya district. The soil mycoflora was evaluated by serial dilution method. Each soil samples were: there is more than one space between weighed of about 10 g, transferred to 100 ml sterilized distilled water and mixed in a

rotary shaker (250 rpm) for 30 min. The soil suspension was diluted from 10-1 to 10-5 and an aliquot of 100 µl from this solution was transferred on to PDA (Potato Dextrose Agar) medium [7]. Chloramphenicol at 0.2 g/ l was added to the medium to avoid any bacterial contamination. The PDA plates were incubated for seven days at 22 ± 2° C. After incubation, the plates were thoroughly examined under stereo-binocular microscope and screened for the incidence of Fusarium moniliformae and were identified based on the morphological, conidial, fruiting bodies and culture characters based on the standard procedures. The pathogen was further subcultured and maintained in test tubes and Petri plates on PDA media and used for further studies.

Calculate the germination percentage of Sorghum bicolor by paper towel method

Paper towel method

One hundred seeds were placed at equidistant on the germination paper presoaked in distilled water and covered with another presoaked paper towel and wrapped with polythene to prevent drying of towels. The paper towels were incubated for ten days at 25 ± 2 C. After incubation, paper towels were unrolled and germinated seeds were counted and represented in percentage. The vigor index was calculated by using the formula VI = (mean root length + mean shoot length) x Germination percentage [1]. The experiment was conducted with four replicates of hundred seeds each and the entire experiment was repeated thrice.

Calculation of germination percentage

The germination percentage of the seeds was calculated using the formula [1].





spores in sterile D/W and centrifuging and finally resuspended in 2-3 ml of sterile D/W. Using this procedure; about 4-5 ml of conidial suspension was obtained. The inoculum was prepared fresh at the time of use for all the inoculation procedures [32].

Inoculation of pathogen to the root of healthy seedlings

Fifteen day old seedlings were dipped in spore suspension (1X106) and incubated for different intervals viz, 2, 4, 6, 8, 12, and 24 hour under darkness at 24±10 C. as the fusarium monilformae invades through root to cause root rot disease.

Maceration technique for observing infected cells

The inoculated seedlings were removed from the spore suspension, fixed in acetic acid (1:3) and processed further for histological observations.

The fixed seedlings were partially macerated in 3% (w/v) sodium hydroxide for 30 minutes at 600c and thoroughly washed in running water for 30 minutes to remove sodium hydroxide [26]. The washed seedlings were transferred to 0.2% (w/v), warm cotton blue and stained for two hours.

RESULTS

Isolation and identification of Pathogen from the Sorghum bicolor L. samples.

Collection of plant material

The local seeds of Mandya district Swathi var. were collected from seed agencies. The infected plant materials were found out and collected showing symptoms of root rot disease from different regions of Mandya district

Infected Sorghum plant in the field Infected sorghum Root

Isolation and identification of F.moniliformae

Isolation of the Pathogen Spores of F.moniliforme





Calculate the germination percentage of Sorghum bicolor by paper towel method.

Fifty randomly selected seeds were placed on moistened paper sheets; ten seeds were arranged per lane. The seeds were rolled carefully to avoid any excess pressure on seeds. This experiment was carried out under growth chamber conditions, at temperatures ranging between 25–28°C, and 12 h light – 12 h darkness. All the seedlings were counted and the percentage of germination was calculated. To find out the seedling vigour, normal

seedlings were taken from the germination test at random. The seedlings gave 92% of the seed germination and process continued for further process.

Hypersensitive Reaction

The inoculate tissues were darkly stained areas were observed. These areas were referred to as hypersensitivity (HR) reacting regions. The HR regions were observed after sixteen hours of inoculation of pathogen.

Preparation of plant material and histological observation

The fixed seedlings were partially macerated in 3% (w/v) sodium hydroxide for 30 minutes at 60º C and thoroughly washed in running tap water for 30 minutes to remove sodium hydroxide. The washed

seedlings were transferred to 0.2% (w/v), warm cotton and stained for two hours.

Histology

Infected root observations in different hours by Maceration technique

Zero hour after inoculation Two hours





Under 10X Under 40X

After Four hours of inoculation where an encasement of the thick hyphae is seen.

Under 10X Under 40X After Six hours after inoculation Increased mycelia growth is seen throughout the host cells.

Under 10X Under 40X





After eight hours of inoculation, the development of conidia and Disintegration of the Host cell resulting in the collapsing of tissues

Under 10X Under 40X

After Sixteen hours of inoculation, there is Hypersensitive Reaction which are darkly stained

Under 10X

Mycelial ramification of the host tissues and complete growth of the mycelia is seen at 24 hr after inoculation with F. moniliforme. The mycelia which is entered intercellular level through the stomata

Discussion

In the present study, an effort has been made to know the process of infection of Fusarium moniliformae in soghum at different interval of times. Fusarium root rot on sorghum typically involves the cortical tissues first and then the vascular tissues of the root [33]. In pearl millet seedlings, the first appearance of hypersensitive reaction occurs in coleoptiles in response to

S.graminicola infection [21]. Hence for histo-pathological studies of S. Bicolor L. both root and coleoptiles were selected. Root rot of sorghum is caused by soil borne fungi, including Fusarium spp., Pythium spp., Macrophomina phaseolina, Colletotrichum graminicola and Periconia circinata [18]. Root rot is generally associated with a complex of these and other fungi and colonization of tissues depends on environmental factors that favour a particular pathogen at a certain time as well as the degree of host predisposition [20]. Early events during plant colonization by F. verticilliodes the transgenic fungus was easily detected on the surfaces of underground organs, such as seeds, roots and hypocotyls. During the first few days after planting, fungus grew both on the surface and





inside lateral roots. Only single hyphae were detected growing along the root, and thickened organelles were observed in hyphae that developed on the surface of lateral roots. These organelles resembled conidia. Hence for histopathological studies of S. Bicolor L. early i.e. three day old seedlings were taken and infected to root and observed the thickened hyphae in the lateral surface of the roots and conidia .F. moniliforme J. Sheld (Sensu late) originally described in 1904 is regarded as one of the major Fusarium spp. that cause root rot, was recently reclassified and the species most commonly found on sorghum has been renamed F. thapsinum Klittich, Leslie, Marasas [6]. In greenhouse and field experiments conducted [30] F. thapsinum was most virulent on sorghum, compared to the other Fusarium spp. tested. These included F. andiyazi and F. verticillioides. Studies have demonstrated that Fusarium spp. infect sorghum rootlets without the development of obvious symptoms until plants reach maturity [10]. At maturity, discolouration of the roots occurs and infected sorghum plants show a reduction in plant growth with poor grain fill and concomitant yield loss when disease is severe. Severe damage to the roots can result in decreased water absorption, nutrient uptake [6] and loss of anchorage through the destruction of older roots [34] This can lead to plants being easily uprooted, lower grain yield, a reduction in drought tolerance and eventually plant death [34] Lesions caused by Fusarium spp. can differ in size, from small circular spots scattered over the roots or stripes that extend over most of the root surface. These are usually red to purple in colour [6] depending on the host genotype, although light brown or black discolouration may be associated with tan plant types. The infection may cause a breakage of the lateral roots from the main root system and infected roots generally lack root hairs (McLaren, 2002). Degeneration of the vascular bundles and pith are rarely caused by Fusarium spp. on roots, but may be caused by secondary or opportunistic organisms that are usually present and can cause the disintegration of tissues [6], e.g. Periconia spp. and other Fusarium spp. [24]. When disintegration of the inner tissues occurs, all that remains is a dry hollow shell that lacks any form of structural integrity [32]

Root rot fungi can be distributed through rain, agricultural equipment, wind and animals (insects in particular) and survive in plants, soil or plant debris either as spores, hyphae or resting structures [33]. Germination of resting cultures or spores is stimulated by root and seed exudates [14] and the pathogens gain access to the roots through natural root wounds or injuries caused by machinery, insects or other causes [6].

Primary infection starts in the cortex tissues and spreads towards the vascular tissues of the root [34]. F. moniliforme J. Sheld (Sensu late) originally described in 1904 and regarded as one of the major Fusarium spp. that cause root rot, was recently reclassified and the species most commonly found on sorghum has been renamed F. thapsinum s [6]. In greenhouse and field experiments conducted by [30] F. thapsinum was most virulent on sorghum, compared to the other Fusarium spp. tested. These included F. andiyazi and F. verticillioide. Studies have demonstrated that Fusarium spp. infect sorghum rootlets without the development of obvious symptoms until plants reach maturity [10]. At maturity, discolouration of the roots occurs and infected sorghum plants show a reduction in plant growth with poor grain fill and concomitant yield loss when disease is severe.

SUMMARY

Sorghum bicolor L. has many nutritional benefits. Its interaction with the pathogen and environment, selection for new early maturing cultivar that can produce high yield and quality seed under prairie growth conditions. In Sorghum bicolor number of investigations have been reported the appearance of pest attacks and diseases that can affect both yield and quality of the plant adversely. It can affect both by biotic and abiotic agents. Fungal plant pathogens have evolved diverse mechanisms for gaining entry through natural openings, wounded cells or by various mechanisms of direct penetration through the outer surface. After the inoculation and observed in sequence intervals of time, it produced spores, germinated spores, conidia, hyphal penetration through the tissue produced germ tube. At the severe infected stage it showed sequential cell death and the causes the root rot finally death of the plant. In this case understanding how fungal pathogens breach the protective barriers that comprise the surface of the host plant will lead to novel strategies for controlling plant diseases.

CONCLUSION

From this study it was clear that Sorghum bicolor L. was found highly susceptible to the Fusarium moniliforme. The sorghum was sprayed and dipped in the fungal solution and the disease incidence was studied with a time interval. It was found that the fungus has the capacity to infect the plant through root causing Root Rot and caused cell death after 7 days of inoculation. Thus F.





moniliforme can be a potential pathogen to the Sorghum bicolor L.

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