Evaluation of the Effect of Combining Fusarium oxysporum and Macrophomina phaseolina on the Incidence of Jute (Corchorus olitorius) Disease

Soil is a complex ecosystem that serves as a home for a diverse range of creatures that coexist with dozens of other species. Due to their diverse life histories, capacity for dispersion and tolerance to heat, microbiomes in natural settings react to changing climatic circumstances in distinct ways (Khamari et al., 2022). Soil microbial biomass is decreased by rising temperatures and cloud cover (Khamari et al., 2022; Rinnan, et al., 2006). Precipitation variations influence the makeup of the microbial community (Castro et al., 2010). Root-associated bacteria have a significant role in determining the plant community, variety and productivity in the soil (Wardle et al., 2004; Wagner et al., 2014). As microorganisms are closely associated with plant roots (Bais et al., 2006), root phenology plays a significant role in rhizosphere interactions and may affect seasonal assemblages of soil microbial groups.

Many economically significant diseases, including root rot, seedling damping-off and vascular wilts, are caused by soil-borne fungal phytopathogens (Lichtenzveig et al., 2006). Jute production is seriously hampered by root rot and wilt diseases brought on by Fusarium species and Macrophomina  phaseolina, which lower yields and worsen fibre quality. To determine if Macrophomina and Fusarium work together or compete in the Odisha environment, research has been designed.

Samples for phytopathogenic fungus isolation were gathered from the INS Farm SOA University’s jute field. In sterile plastic bags, suspect stems and roots that seemed to have Fusarium wilt and Charcoal Rot symptoms were collected. The removed plant components from the gathered plant samples, such as twigs, bark, stems and roots, were cleaned with sterile water before being surface sterilised with 1% sodium hypochlorite. After that, samples of the roots and stems were divided into 1 cm-long pieces. The researchers used a 7-day-old fresh culture that had been incubated on PDA with 50 mg/L of streptomycin sulphate for 3 to 7 days after being sterilised (Khamari et al., 2019).
 
In vitro investigation of macrophomina and fusarium’s interactions
 
A dual culture approach was used to explore the interaction between Macrophomina and Fusarium, retaining a 5 mm mycelia disc of each pathogen on the opposite side of the petriplates in three replications. A collection of distinct test fungi was kept as a control. Every day, observations were made to examine how they interacted with one another. A tiny piece of mycelia from the area of contact was placed on a slide and it was examined under a microscope.
 
In vitro investigation of disease complexes
 
An in vitro experiment was carried out to investigate the individual and combined effects of Macrophomina and Fusarium on jute stem. 250 ml of conical flask were filled with 150 ml of potato dextrose broth. Two healthy sesame cut stems, each measuring 10 cm in length and 6.5 mm in diameter, were retained within the flask and were carefully sealed with non-absorbent cotton. 16 conical flasks were also included in the experiment. Every flask was autoclaved.

After chilling, a 5 mm disc of Macrophomina was added to the flask, followed by a second inoculation of Fusarium, a third inoculation of both Macrophomina and Fusarium and a fourth flask that was left uninoculated, serving as the control. The replications were kept at four. After that, these flasks were left alone and routine observations were made at 2, 4, 6 and 14 days following inoculation. 
 
In vivo investigation of disease complexes
 
The standard protocal recommended by Khamari et al. (2019) used to propagate Macrophomina phaseolina, Fusarium oxysporum and both, M. phaseolina + F. oxysporum,  in sand cone media before being injected at a rate of 2 g/kg soil, experimentation were conducted in plastic pots. One treatment was kept uninoculated as a control. All pots were kept in a completely randomized pattern and seeded with J.R.C-212 (Sonali) seed variety. Five replications of each treatment were carried out and observations were made. Disease incidence data were kept daily and during germination and statistical analysis was done to determine how therapy affected certain plant development parameters.

Dual culture technique
 
The pathogens multiplied and mixed with one another. Macrophomina and Fusarium did not exhibit any aggressive behaviour against one another. These findings are similar with Khamari et al., (2017) and it was also shown that Macrophomina spread faster than Fusarium, covering the majority of the plate. When both diseases were seen together under a microscope, they coexisted.
 
In vitro cut stem inoculation method
 
After applying Macrophomina, Fusarium and a combination of both to the sesame stem cuttings, observations were made at 2, 4, 6 and 14 days. There was a thin layer of mycelium covering the medium, which was white in colour. After two days of inoculation, the incidence of Macrophomina and Fusarium was low and the incidence of the two together was moderate. After 4 days following inoculation, the mycelium in the presence of Macrophomina, Fusarium and Macrophomina + Fusarium, respectively, changes to grey, white and white+grey (Table 1).



The mycelium was discovered to have spread up to half the length of the stem together with complete medium coverage by Macrophomina species and only partial medium coverage by Fusarium species without stem infection and a combination of both showed complete moderate coverage with a quarter of stem infection. Due to the inoculation of Macrophomina, the colour gradually changed to a dark grey covering the entire medium and the stem. Contrarily, Fusarium inoculation resulted in the colour turning creamy white and encompassing the entire surface and a section of the stem. Both treatments combined to finally give the colour a greyish tone after six days. After a 14-day inoculation period, we found that Macrophomina, Fusarium and pathogens alone or in combination covered the entire medium and the entire stem, resulting in a change in color to black, milky and greyish. These results are consistent with the study by (Khamari et al., 2017). According to the inoculation research, the stem’s colour changed from white to grey to black at various inoculation days when exposed to Macrophomina, Fusarium and the combination of Macrophomina+Fusarium.
 
In vivo investigation of disease complexes
 
The soil inoculation study found that, under controlled conditions, inoculating with Macrophomina and Fusarium resulted in disease incidence rising to 78.00% and 20.00% seed germination, followed by Macrophomina alone (seed germination of 30% and disease incidence of 66%) and Fusarium recording 56% seed germination and 54% disease incidence. The control pots, on the other hand, had 78% of the seeds germinate without any pathogen inoculation. Khamari et al. (2017), who carried out the same experiment on sesame, published similar results, which are supported by these findings.

In comparison to Fusarium, Macrophomina activity was shown to be faster in terms of dual culture method and soil inoculation experiment. It has been established that Macrophomina grows more quickly than Fusarium. However, the combination of Macrophomina and Fusarium had no antagonistic effects and it was discovered that both, when present together, made the illness worse than when each was present alone (Table 2).



Numerous researchers have already examined the combined impact of Macrophomina and Fusarium on numerous crops. Noted that Fusarium verticilloides and M. phaseolina were in charge of collar rot, seedling rot and other infections in okra. Additionally, they noticed that infected seeds resulted in less seed germination and pre- and post-emergence mortality. Brinjal growth is severely reduced by M. phaseolina +Fusarium oxysporum and carbendazim considerably reduced the fungal complex (Haseeb and Archana, 2009).

The study provides significant fresh knowledge about the interaction between Fusarium oxysporum and Macrophomina phaseolina on the frequency of disease in jute. It further indicates that there are no antagonistic effects between the two funguses. Rather, it was discovered that their combined presence made the infection worse than it would have if either fungus had worked alone.

The results emphasize the deep relationships between plant roots and microorganisms, the intricacy of soil ecosystems, and the difficulties in controlling soil-borne fungal phytopathogens, which cause economically important diseases like root rot and wilt in crops like jute. Thus, the study highlights the need for more investigation to gain a deeper understanding of these relationships and create practical plans for managing diseases in jute farming.

Authors thankfully acknowledge assistance and Lab facilities from Department of plant pathology, IAS, Siksha ’o’ Anusandha University, Bhubhabeswar, India.

Authors declare that they have no competing of interests.