Alternaria leaf spot is a serious problem in both the main crop (i.e. harvest stage) and seed crop of Brassica vegetables (Prasad and Vishnuvat, 2006). It causes huge economic losses to the farmers by attacking the crops at seedling stage, vegetative stage, curding/heading stage (economic harvest) and seed crop in case of vegetable Brassicas (Singh and Kalia, 2021). Since, some of the members of Brassica coenospecies and wild species of Brassicaceae have been reported as resistant against A. brassicae (Sharma et al. 2002) and A. brassicicola (Westman and Dickson 1999) through in vivo and in vitro screening studies. However, there was need to devise a proper protocol from isolation of prevalent isolate of the pathogen (i.e. A. brassicicola) from the region to categorize the species using a proper scale. Further, almost screening practices were confined to the cotyledon stage (Meena et al. 2016) and seedling or young stage of the plants (Sharma et al. 2002; 2004) while it is important to know the level of resistance at adult stage. Sharma et al. (2004) reported that the level of resistance against A. brassicicola in cauliflower decreased from seedling to young and adult stages. Similar cases were also reported in case of downy mildew (Hyaloperonospora parasitica) in broccoli (B. oleracea var. italica L.) (Dickson and Petzoldt, 1993) and cauliflower (Singh et al. 2013). Thus, the screening of wild Brassicas at full-grown adult stage was more appropriate for age-independent resistant sources for breeding use unless the resistance is known to be stable across the plant age.
The A. brassicicola isolate from cauliflower was used to screen the wild Brassica species. During the survey of the prevalent species of Alternaria in cauliflower in 30 random samples from leaf, curds and siliqua in Delhi region, it was observed that A. brassicicola was the most prevalent while infection of A. brassicae and A. alternata. The identification of the species was done on the basis of conidia morphology and sequence analysis. A similar approach was also followed by Sharma et al. (2002) while screening Brassica coenospecies against A. brassicae. Since, variation of host response against the pathogen also depends upon the isolates. Shi et al. (2021) tested 175 isolates of Alternaria and tested their pathogenicity by detached leaves on Chinese cabbage with the disease incidence and disease index ranging from 73.3% to 100.0%.
Further, leaf epicuticular wax is reported to have role in plant defence particularly by creating a hybrophobic surface that decrease the reaction of water-borne inoculum, reducing germination of conidia and causing fewer germ tubes to be produced (Conn and Tewari, 1989). Thus, in present study, the wax layer of the leaves was kept intact while screening the genotypes.
Since, leaf colour and turgidity are important indicators for freshness, thus, both were monitored during the screening process. A moist cotton ball (5% sucrose solution) was effective to retain the leaf of most of the species turgid till 6th day of inoculation, however, the chlorosis was independent of this supplement. The A. brassicicola is a necrotroph and it can grow on yellow or dead leaf tissues. Thus, reaction of the species was not affected by the change in colour, however, occurrence of other saprophytes on decaying leaves influence lesion progression and observations.
Sharma et al. (2002) suggested for a cumulative score from observation on incubation period, disease rating and lesion size. While, Sharma et al. (2004; 2012), Singh et al. (2008), and Meena et al. (20016) used disease rating or PDI for categorization of genotypes in different disease reaction groups. Significant correlation between disease rating and PDI (r2= 0.903) and cumulative disease score (r2=0.928) indicates for the effectiveness of different disease parameters.
Change in lesion size is an important indicator of genotype response to the host-pathogen (Sharma et al. 2002; 2004). Significant variation among the species for increase of the lesion diameter on 6th day post-inoculation indicates that these species have some resistance mechanism that is activated following infection and inhibits or slow down the growth of fungus as also indicated by Sharma et al. (2002) while screening Brassica coenospecies against A. brassicae.
The appearance of observable symptoms after inoculation of leaves with A. brassicicola is an important indicator at the initial phase of host-pathogen interaction. There was significant variation among species for the incubation period during in vitro challenge inoculation which could be due to role of defence substances such as camalexim produced during the germination and establishment of the pathogen (Browne et al. 1991).
Vishwanath and Kolte (1999) reported that detached true leaf inoculation as the most efficient and reliable method for host screening for resistance to Alternaria brassicae in rapeseed mustard. It has no interference from local growing conditions (Hong et al., 1996; Scholze, 2002; Shrestha et al. 2005), the suboptimal developmental stage upon pathogen incidence, or presence of other strains and or pathogens under natural epidemiological conditions (Michereff et al. 2012) and length of growing period (Sharma et al. 2002). Nowakowska et al. (2016) also in the view that this procedure is equally effective as of seedling stage screening to discriminate resistant and susceptible genotypes. It has been validated in different crops i.e. oilseed Brassicas (Vishawanath et al., 1999; Meena et al., 2016) and wild Brassicas (Sharma et al. 2002), cabbage and cauliflower (Sharma et al., 2004). This reduces the chance of variation in disease rating in field conditions which could arise due to differences in growing and infection conditions. Further, we suggest the detached leaf inoculation method for screening wild Brassica species for reasons: (i) it is always better to do primary screening against a pathogen which have a wide host range in fully controlled condition, (ii) it is an effective way to screen large germplasm with limited resources available, (iii) avoid contamination of germplasm since it is a seed-borne disease, (iv) it avoids the escape of pathogen in a new environment, (v) field screening requires special arrangements to contain the pathogen, (vi) growing wild species in field condition may disastrous since there are possibilities to escape and became a weed or may contaminate/pollinate the common Brassica crops or minor Brassica weed species in the region and (v) Doula et al. (2006) reported a significant positive correlation between Alternaria leaf spot disease rating in detached leaf test and seedling test.
Only moderate resistance was reported in cauliflower by Nowakiwaska et al. (2016), in red cabbage (PI291998) and cauliflower (PI291565 and PI441510) and broccoli (IHRGRU04, 003571 and IHRGRU04,004712), however, dealing with such devastating necrotrophic parthogen, it there is urgent need to find stable and robust resistance source(s). Besides, in vegetable Brassicas, the most devastating impact is during their economic maturity (i.e. edible parts) since infection during this phase directly affect the market price and consumer acceptance. No robust donor sources reported so far in the primary gene pool (GP1) of cultivated Brassicas, hence it is imperative to search the available germplasm of wild relatives (GP2 and GP3) of Brassicaceae for use in resistance breeding. Buchwaldt and Green (1992) screened 19 species of Brassicaceae against A. brassicase and its potential phytotoxin destruxin. Later, Sharma et al. (2002) screened 38 species of Brassica coenospecies against A. brassicae and identified B. desnottessi, Camelia sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretica, D. erucoides and Erucastrum gallicum as resistant. In present study, Capsella bursa pastoris (early) and Capsella bursa pastoris (late) showed symptomless resistance during all three years. The resistance in Camelia sativa and D. erucoides against A. brassicae (Sharma et al. 2002) and A. brassicicola as observed in present study indicates their broader prospect in resistance breeding.
Symptomless resistance was observed in both genotypes of Capsella bursa-pastoris during 2019-20, 2020-21 and 2021-22 years. The observations are in agreement with the findings of Conn et al. (1988) and Buchwaldt and Green (1992) who had reported resistance in C. bursa-pastoris, Camelina sativa, D. erucoides, Sinapis alba and Thilaspi arvense against A. brassiciae. Sharma et al. (2002) also reported resistance in wild relatives of Brassicas namely B. maurorum (Chrungu et al., 1999), B. desnottesii, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides and Erucastrum gallicum. Westman and Dickson (1999) identified Camelia sativa and Capsella bursa-pastoris as highly resistant to Alternariabrassicicola. Few members of Brassica coenospecies viz., C. sativa, S. alba and D. berthautti were also have been found to be resistant against A. brassicae, to another major pathogen of Alternaria leaf spot (Sharma et al. 2002). The durable resistance in these wild species to Alternaria leaf spot could be attributed to the co-evolution of resistance since these species remain in wild or weed state and adapted to prevalent biotic and abiotic stresses. Significant correlation among the observations from all three years indicates the robustness of the screening procedure. A poor correlation between leaf colour and disease rating supports the necrotrophic habit of the A. brassicicola which multiplies on dead tissues also.