Identification based on rDNA sequence analysis
Nucleotide sequence analyses of ITS and beta tubulin regions of rDNA were carried out to characterize the S. rolfsii genetically. PCR amplified products of ITS and beta tubulin rDNA yielded approximately 527 and 448 bp amplicon sizes, respectively, when visualized at 1% agarose gel (Fig. 1). Nucleotide sequences of ITS and beta tubulin amplified rDNA were deposited in GenBank under the accession No. MT573510 and MN736404, respectively.
In vitro antifungal bioassays
In vitro effect of methanolic leaf extract against the growth of S. rolfsii was illustrated in Fig. 2. It clearly indicated that all the concentrations significantly (P ≤ 0.05) reduced the fungal biomass than the control. Different concentrations were found to reduce the fungal biomass by 86–90% over control treatment. These findings are in the favor of work of Obongoya et al. (2010) who evaluated the antifungal efficacy of Neem extract against Fusarium oxysporum with promising results. Generally, plant extract efficacy was influenced by the level of concentrations used in experimental work; however, in the present study, the increase in concentration did not result in a parallel decline in mycelial growth of the pathogen. Likewise, Akaeze and Aduramigba-Modupe (2017) checked in vitro efficacy of Neem plant extracts against F. oxysporum, where all the concentrations of methanolic extract were found almost equally effective against F. oxysporum.
Pot trial
After 15 days of sowing, the highest seedling mortality (42%) was recorded in positive control, which increased to 56% after 30 days of sowing. Mancozeb application was highly effective in reducing plant mortality. In this fungicidal treatment, the plant mortality was just 3% after 15 days of sowing that was increased to 28% after 30 days of sowing. The lowest concentration (1%) of dry leaf biomass (DLB) did not show any remarkable effect as there were 33 and 49% disease incidences after 15 and 30 days of sowing, respectively. However, 2 and 3% DLB significantly reduced disease incidences over positive control both after 15 and 30 days of sowing (Fig. 3).
In pot trials, the collar rot of chickpea was managed through the use of a fungicide namely, mancozeb and Neem dry leaf biomass. Disease highly occurred in positive control, whereas negative control was disease free. The disease incidence was reduced significantly in Neem dry leaf biomass and mancozeb-treated soils. The fungicide gave better results than the dry leaf biomass. There are reports that fungicides are highly effective for the control of target fungus S. rolfsii (Shirsole et al. 2019). Mancozeb proved to be a broad-spectrum fungicide effective against downy mildew, collar rot, foot rot, damping-off, stem rot, root rot, southern blight, bulb rot, and wilt diseases caused by Lagenaria siceraria, Alternaria solani, Rhizoctonia solani, Sclerotium rolfsii, R. bataticola, and F. oxysporum (Majumder et al. 2016; Gowari et al. 2017; Bagri et al. 2019; Michel-Aceves et al. 2019 and Vani et al. 2019). Rather et al. (2012) concluded that many fungicides like tebuconazole, penconazole, vitavax, hexaconazole, thiophanate methyl, and mancozeb were effective against S. rolfsii. Similarly, Madhavi and Bhattiprolu (2011) reported that in field experiments, mancozeb was highly effective for the control of S. rolfsii responsible of dry root rot in chilies. In a previous study, it was also reported that F. oxysporum isolated from the cucumber-infected seeds was completely inhibited even at lower concentrations of mancozeb (Sultana and Ghaffar 2013).
All the concentrations of Neem dry leaf biomass significantly reduced the pathogen growth and disease incidence over control. In a previous study, Obongoya et al. (2010) revealed that F. oxysporum, the pathogen of yellow disease, can effectively be managed through the botanicals isolated from Neem plant. In their study, soil amendment with Neem was also observed to be effective in enhancing the plant size and seed germination with minimum seedling mortality by F. oxysporum. Ezeonu et al. (2018) also gave similar report regarding the effectiveness of Neem seed, bark, and leaf ethanolic extracts against fungal pathogens namely, Aspergillus oryzae, A. niger, Rhizopus stolonifer, A. ochraceus, and Lasiodiplodia theobromae responsible for rot diseases in cocoyam and yam plants. Obtained results agree with the findings of Ali et al. (2017b) who discovered that nano emulsions of Neem represent excellent antifungal activity against many phytopathogenic fungi such as follows: S. rolfsii and Rhizoctonia solani that cause collar rot, damping-off, wilting, and dry root rot under field conditions.
The effect of A. indica DLB and fungicide on the shoot growth of chickpea is shown in Fig. 4. There was insignificant difference in shoot length between positive and negative control treatments. Applications of 1, 2, and 3% DLB of A. indica and mancozeb significantly reduced shoot length over negative and positive controls. In contrast, shoot fresh weight at 1 and 2% DLB amended treatments showed significant increases over positive control, whereas in mancozeb treatment, it was significantly lower than negative control. Shoot dry biomass in 1% amendment was at par with negative control. However, as the concentration of DLB was increased, it adversely affected the production of shoot biomass.
The effect of DLB and fungicide on the root growth is shown in Fig. 5. Significant reductions in positive and negative control root lengths with enhanced dry root weights were recorded in comparison to mancozeb application. The root lengths and dry biomasses increased significantly in 1, 2, and 3% concentrations of DLB over positive control. The effect of 1% DLB was more pronounced than the effect of higher concentrations of 2 and 3%. In general, increase in DLB dose resulted in reduction of root growth parameters.