While observing the experiment, it was noticed that there was root induction in some of the inoculated bulbs, and the histopathology of roots induced from the Disc Inoculation Method was studied, as roots induced were more prominent. In this method, we noticed the root induction in the inoculated bulb at three DAI. The inoculated bulbs were planted in pots with sterilized soil to observe the germination, and after ten days of seeding the bulbs, it was observed that only F. acutatum infected bulbs (Fig. 6b) sprouted after 12 days. While there was no sprouting in the case of bulbs inoculated with F. falciforme, rather F. falciforme infected bulbs were completely rotten (Fig. 6c). The above roots from F. acutatum inoculated bulbs and control bulbs (Fig. 6a) were subjected to histological studies at 40 DAI, which indicated damages to epidermal, cortex tissues, and xylem tissues of the roots appeared in the form of dark brown color (Fig. 6e). However, roots from un-inoculated bulbs had no damage or browning (Fig. 6d) in the vascular bundles in the xylem and cortical tissues.
Standardization of laboratory-based reliable and reproducible artificial inoculation methods is of fundamental requirement for evaluating pathogen-host interactions, epidemiological studies, germplasm screening for disease resistance, and host responses to pathogen. Therefore, the current study was conceived to optimize the artificial challenge inoculation methods to induce FBR in onion bulbs of the 'Bhima Super' variety under Indian climatic conditions. Although previous studies have described the methods of challenge inoculation for FBR in 'onion seedlings', but the studies on inoculation methods on 'onion bulb' are inadequate. It is interesting to note if a particular Fusarium strain equally affects both 'onion seedlings' and the 'onion bulbs' of the same cultivar. Earlier, Taylor et al. reported that the particular F. oxysporum isolate with high virulence would produce similar effects on 'bulbs' and 'seedlings' of the same cultivar. Subsequently, contrary reports have shown that the virulence levels of pathogens in seedlings and in bulbs differ significantly, indicating that the disease outcome is driven by phenological stages of onion. The Fusarium isolates that were virulent on seedlings were sometimes not virulent on bulbs and vice versa, pointing to the specialization of isolates for the host phenological stage. Therefore, the challenge inoculation method developed for 'onion seedlings' may not work for 'onion bulbs'. Furthermore, the challenge inoculation methods described for one geographic region may not be reproducible in a different region because the growth of different Fusarium sp., their symptomatology, and the epidemiology are reported to be influenced by environmental conditions. For a country like India, which holds a rich diversity of onion germplasm, the development of the in vitro method of bulb inoculation is needed for the systematic screening of available germplasm for FBR resistance. Therefore, we devised five different challenge inoculation methods for artificially inducing the FBR in onion bulbs. The well-established knowledge that the Fusarium infection primarily occurs through direct penetration of the 'basal stem plate' or the mechanical injury near the basal portion of bulb scales or the natural openings described earlier by Cramer and coworkers formed the basis for devising the five different inoculation methods. Additionally, the key aspects of Fusarium biology, such as conidia production in 10-15-day-old cultures and the inoculum density required to establish infection, as reported by Galvan et al., were also considered while formulating these methods.
The well-characterized basal rot pathogenic cultures of F. acutatum (OGRDFW4) and F. falciforme (OGRDFW7) were used to compare the efficacy of methods for different strains of Fusarium described elsewhere. The inoculum density is the determining factor in initiating the infection by the Fusarium strain for the development of basal rot on onion bulbs. Previous studieshave established a direct correlation between the inoculum density of Fusarium oxysporum f. sp. cepae and the incidence of Fusarium basal rot (FBR) in onion seedlings. Higher inoculum densities of Fusarium spp. have been shown to directly impact the final disease incidence and accelerate the onset of early symptoms. In our study, a spore concentration of 10⁶ conidia/mL was used across all five methods, based on the effectiveness of this conidial concentration in our earlier study and also based on the reported conidial concentrations used in studies by other groups. Using a pre-defined conidial concentration ensures challenging the bulbs with uniform pathogen pressure applied across treatments, allowing for a reliable comparison across different methods used and also across other related studies.
Among the five methods evaluated in the current study, the method involving 'cotton swab impregnated with spore suspension', resulted in typical symptoms of mycelial growth and necrosis observed within seven DAI, and rotting observed in 11 days, with complete rotting of the bulbs in 15 days for both Fusarium species. The affected surface area and volume of rotting area was higher for F. falciforme compared to F. acutatum which indicated F. falciforme being more aggressive than F. acutatum in later stages of infection. The higher aggressiveness of F. falciforme than F. acutatum is in disagreement with previous report of Bhat et al. reporting a very high virulence of F. acutatum isolates whereas a range of virulence was noticed for tested F. falciforme isolates. Still, the two strains, F. acutatum and F. falciforme, are more prevalent in Indian climatic conditions in contrary to F. oxysporum being the most prevalent species for FBR in many other locations world-wide.
The cotton swab method (Cotton swab impregnated with spore suspension) did not involve any mechanical injury made to the bulb and hence mimicked the natural infection conditions by far extent, still produced best results in terms of larger affected surface area, larger affected bulb tissue volume and overall rotting percentage and higher disease rating score. The success of this sort of cotton swab method has been previously demonstrated by Iftikhar et al. for screening of wheat cultivars against Bipolaris sorokiniana by using a moist cotton swab in test tubes containing seeds. The authors evaluated different methods and adjudged the 'cotton swabbing' method as best among the five methods evaluated to establish the Bipolaris sorokiniana (Cochliobolus sativus) infection in wheat seedlings. Therefore, we also tried the cotton swab method and found that the infection was achieved within seven DAI leading to the maximum rotting percentage (92% for F. falciforme and 56% for F. acutatum). This method is regarded superior as this method does not involve any mechanical injury to the bulb and such fungal inoculation studies using cotton swab method are not reported previously for onion. Moreover, this method is rapid, easy to execute and cost-effective, and might be useful in screening FBR resistance in large numbers of onion genotypes. The method 3 involving 'Pinprick at basal plate and dipping in spore suspension plus Cotton swab' also showed comparably higher rotting percentage (94%), high volume of affected bulb tissue (69 cm) and disease rating of '9' for F. falciforme and is therefore equally good method for challenge inoculation of bulb.
The pinpricking at the basal plate and dipping in spore suspension plus cotton swabbing, caused almost complete rotting (94%) in 13 days. This combination method was tried for the first time to compare the differences in virulence caused by the single and combination methods. The more rotting caused by this method might be attributed to a longer period of mechanically injured bulbs being associated with inoculum. This attempted method was almost at par with the 'cotton swabbing method'. However, we rate this method inferior to 'cotton swab method' because it involves a direct mechanical injury (pinpricking) to the bulbs which renders the direct entry of pathogen to the bulb interior tissues and therefore does not represent natural infection conditions. The FBR bulb pathogenicity assay by creating a physical wounding to the bulb has earlier been attempted by Le et al., and Wang et al. for assessment of infection potential and also the VOCs (Volatile Organic Compounds) and mycotoxin profiling of inoculated bulbs with different Fusarium species. The mycotoxin like beauvericin (BEA) are produced by certain species of Fusarium that can have association with its virulence or host/phenological stage specificity. A higher amount of mycotoxin beauvericin (BEA) was detected in the seedlings infected with F. oxysporum isolates but not in the seedlings infected with F. solani suggesting a differential mycotoxin profile of seedling for different Fusarium species. Similarly, authors also noted quantitatively higher and compositional diverse mycotoxin levels in onion bulbs compared to the seedlings after three weeks of inoculation. Such Fusarium mycotoxins like beauvericin (BEA), fumonisins (FBs), moniliformin (MON), and fusarin can suppress plant defenses while the production of VOCs (Volatile Organic Compounds) can modulate plant stress responses and microbial interactions, aiding to pathogen spread.
In Method 2, pinprick at the basal plate and dipping in spore suspension, both Fusarium species took seven days to produce mycelium like in the cotton swabbing method. F. falciforme was more aggressive leading to complete rotting of the bulbs at 13 DAI, whereas in F. acutatum only 68% rotting area was recorded. Additionally, the oozing of watery content was observed (13 DAI) in the case of F. falciforme, which might be due to the complete maceration of tissues of the infected bulbs. Previous studies have reported different inoculation methods, including dipping bulbs in spore suspension, making holes in bulbs, and wounding the basal plate before dipping in spore suspension. The results obtained are in line with that of Ghanbarzadeh et al. and Sogoba et al. who made holes or perforations on the bulbs for introducing the suspension of F. oxysporum, F. solani, F. proliferatum, F. redolens, F. falciforme and F. acutatum for evaluating symptom developments and pathogenic potential of Fusarium strains. As compared to the cotton swab method, in this method, the rotting area was less in the case of F. acutatum, while in F. falciforme whole basal plate was rotten in 13 DAI. Our results are not in line with those of Toit et al. who reported the wounding of basal plate and dipping in spore suspension did not achieve clear FBR symptoms with F. proliferatum, rather developed a salmon-pink discoloration of the outer scales in 3 weeks. However, in our experiments, we could induce the basal rot symptoms and complete rotting of the bulbs in 13 days.
In the disk inoculation method 4, in F. acutatum inoculated bulbs, mycelial growth was observed as early as three DAI, while F. falciforme inoculated bulbs showed mycelial growth after five DAI. The early development of white mycelial growth on the bulb may be attributed to the direct contact of actively growing mycelial disk to onion bulb basal plate, rendering an opportunity for to pathogen to use nutrients from the bulb for its own growth and easily penetrating the bulb tissue through its cellulolytic and pectinolytic activity. Although this study was not aimed to explore the mechanistic details of Fusarium invasion of onion bulbs, the extracellular hydrolytic enzymes, collectively called cell wall-degrading enzymes (CWDE) are known to play indispensable role in Fusarium pathogenesis of plant tissue as described earlier. Therefore, it would be interesting to compare the variable activity of cell wall-degrading enzymes (CWDE) in highly virulent F. falciforme and its comparison to F. acutatum strains used in our study. Compared to other methods, there was no complete rotting of the bulbs in this method, however, the disease symptoms developed early. The rotting area of F. falciforme was more than F. acutatum. This confirmed the infection in the experimental bulbs. The results obtained are in agreement with Tirado-Ramirez et al., who reported that the F. falciforme is more aggressive on the onion bulb than F. oxysporum. However, some isolated studies have reported contradictory findings, indicating that mycelial inoculation did not successfully induce FBR in bulbs, which might be due to disease escape due to environmental, physiological, or spatial factors that prevent infection. It is also seen that some time the beneficial microbes inherently present in the onion bulb as a part of plant's microbiome may outcompete or inhibit the pathogen. Although, the efficacy of disk inoculation methods should not be directly compared with other methods because of the inoculum load applied could not be compared with other methods. Maintaining uniform propagule pressure was not possible with this inoculation method (disk inoculation) due to the variable growth of fungal mycelium and sporulation on nutrient-rich PDA media, still, we achieved the desired infection in the bulbs. Shin et al. also showed disk inoculation of F. commune, F. oxysporum, and F. proliferatum on the basal plate producing mycelial growth in 8 days.
In the injection of spore suspension (method 5), mycelial growth was observed at seven DAI, which might be due to longer time taken for the spore germination and infection. Though the reduced rate of infection caused slow infection, the rotting was almost at par with method 4. This is a common method for inoculation to prove the pathogenicity of FBR caused by F. falciforme, F. acutatum, F. solani and F. oxysporum and has been used by several workers. The disease symptoms tend to develop slower than with other methods. Our results in this study are in agreement with Mandal and Cramer, who reported that conidial inoculation with the removal of basal plate top causes less incidence of FBR. The cutting of basal plate and injecting spores, and their interactions were the determinants of successful disease development causing 82% rotting. However, this method is difficult to apply for screening a large number of bulbs, being tedious and requiring more care and manpower to carry out the work as compared to other methods.
In our results, we found all five methods were effective in causing infection. In comparison, Method 1 and Method 3 were found best as both caused early infection with less time and labour. These methods would be feasible for selecting/ screening FBR-resistant bulbs under field conditions. The result of this study confirms the negative assertion that disk inoculation is not effective in selecting FBR-resistant bulbs. A higher disease pressure had been observed using mycelium inoculation as compared to the spore inoculation methods, it has been recommended to use conidial suspension as mycelium inoculation is hard to quantify, and a standardized spore concentration is difficult to obtain. Also in this method, the initial fungal establishment for causing infection early under in vitro conditions is easy, however keeping inoculated bulbs open in a greenhouse or field would be difficult to get the infection. This exposure of the PDA plugs to direct sunlight could dry the inoculation surface prematurely before successful pathogen invasion or due to desiccation of the inoculated surface and PDA plugs delay the infection under field/greenhouse. So, the disk inoculation method is not practical for screening many genotypes.
In the disc inoculation method, we noticed the prominent and consistent root induction in the inoculated bulbs of both species at three DAI, however number of roots was more in F. acutatum inoculated bulbs than F. falciforme, and invariably, there was no root induction in control. When these infected bulbs were planted in pots under controlled conditions, it was observed that F. acutatum infected bulbs sprouted in 12 days while F. falciforme infected bulbs failed to sprout, rather all bulbs were rotten in soil which may be attributed to higher magnitude of disease by this species. In bulbs inoculated with F. acutatum slower disease progression might have led to profuse rooting and delayed rotting. The regeneration of roots in bulbs infected with F. acutatum might be due to the possible activation of root-inducing hormones. Observation of roots' histopathology indicated that damaged tissues showed a dark brown color in roots from F. acutatum infected bulbs. The observed brownish color in damaged root tissues might be due to the degradation of phenolic compounds into less simple compounds and their absorption by the cell wall of the damaged root tissues. The changes of the infected root tissue into a brownish or blackish color indicate pathogen establishment and damage caused by F. acutatum inoculation. Our results agree with reports of Nasir et al. and Purwati et al. who recorded similar findings in their studies.
Additionally, we noticed the induction of roots in the F. acutatum inoculated bulb for methods 2 (Pinprick at the basal plate and dipping in spore suspension) and method 3 (Pinprick at basal plate and dipping in spore suspension plus Cotton swab), however the same methods with F. falcifome inoculation did not result in root induction. This implies that both the Fusarium strains used in this study were pathogenic, resulting in progressive rotting of the bulb, but the strain F. acutatum might exhibit root inducing hormone-modulating abilities as some fungal pathogens, such as Fusarium spp. are known to produce auxins as part of their interaction with plant hosts. This might initially stimulate root formation before the pathogenic phase sets in or else it could be an innate plant response to the fungal invasion where the 'bulb might induce itself to regenerate root tissue in reaction to fungal attack. Although, this aspect of pathogen-bulb tissue interaction was not explored in detail, as we intended to optimize the method for artificial bulb inoculation, it would be interesting to explore this aspect of host-pathogen interaction. As a preliminary microscopic examination of the induced roots in F. acutatum inoculated plants, we noticed that the F. acutatum strain can colonize epidermal, cortex and xylem tissue (Fig. 5) to impair the water transport and structural tissues aggravating the symptoms like tissue rotting.
Based on the experiments conducted here, the recommended protocol to determine the inoculation methods for the creation of artificial FBR infection would include the following steps: Protocol 1: (i) Surface sterilization of bulbs by wiping with 70% ethanol (ii) Removing dead roots and dried outer scales and making a thin cut (~ 0.25-0.30 mm) in basal plate (iii) Dipping the cotton swabs in ~ 2 ml of spores suspension (1 × 10 spores/mL) (iv) Keeping swabs on the basal plate and temporarily holding with cello tape (v) Placing in polythene bag (vi) Incubation of inoculated bulbs under alternating light conditions (12 h light and 12 h dark) at 27 ± 2 °C. This was followed by Protocol 2: (i) Surface sterilization of bulbs by wiping with 70% ethanol (ii) Removing dead roots and dried outer scales (iii) Cutting of thin layer (~ 0.25-0.30 mm) of the basal plate (iv) Making holes (approximately 5 mm depth) on the basal plate and dipping in 10 mL suspension (1 × 10 spores/mL) for 2 h. (v) Placing in polythene bag (vi) Incubation of inoculated bulbs under alternating light conditions (12 h light and 12 h dark) at 27 ± 2 °C. Bulbs were monitored daily to observe the expression of symptoms.
It is a challenge to develop an inoculation protocol to deliver consistent disease pressure that will produce repeatable results. Virulent cultures with morphological and molecular analyses of pathogen cultures of Fusarium falciforme and Fusarium acutatum were obtained. Five inoculation methods viz., Cotton swab impregnated with spore suspension; Pinprick at the basal plate and dipping in spore suspension; and a combination of pinprick at the basal plate and dipping in spore suspension plus cotton swab; disk inoculation, and injection of spore suspension, were compared and a high throughput methods were standardized. Two protocols, i.e., 'The cotton swab' method and 'Pinprick at the basal plate, and dipping in spore suspension' were found best, and these standardized protocols are being recommended to be used by the stakeholder for creating artificial FBR infection. Between two Fusarium spp., the FBR magnitude was higher from F. falciforme indicating aggressiveness than F. acutatum. The protocols suggested in this study would be useful for finding sources of resistance and implementing sustainable management strategies for these pathogens.