The 32 isolates of A. brassicae, 20 isolates of A. brassicicola and 3 isolates of A. alternate originating from diverse regions of North-West India, where Alternaria blight is highly prevalent, were investigated for their pathogenicity on various host species. The disease reaction varied with necrotic lesions of 0.4-7.5mm, mostly having brown to black coloured lesions. The isolates caused moderate to severe chlorosis on B. rapa varieties (YSH-401 and Pusa gold) as compared to the B. juncea varieties (Varuna, Rohini, Kranti and PHR-01). The mean disease index varied from 2.22±1.17 to 7.32±0.67 in A. brassicae isolates, 2.38±0.92 to 5.89±0.89 in A. brassicicola isolates and 2.49±0.97 to 5.87±0.91 in A. alternate isolates. Out of seven A. brassicae isolates that tested positive for non-aggressiveness on specific host species, five isolates, on prior inoculation, induced tolerance to highly aggressive strains of A. brassicae in the respective B. juncea and B. rapa varieties. The study thus opens up the possibility of deploying attenuated virulence as naturally occurring biological control for induction of systemic resistance/tolerance in Brassicas against A. brassicae, one of the most destructive fungal pathogen.

Keywords: alternaria blight, oilseed brassica, pathogenicity, attenuated virulence, systemic resistance

PDA, potato dextrose agar; SA, salicylic acid; JA, jasmonic acid; ISR, induced systemic resistance; PR, pathogenesis-related; DI, disease index

Commercial cultivars of Citrus spp. (Rutaceae), including oranges, mandarins or tangerines, grapefruits, lemons, and limes are the most widely produced fruits in the world. China, Brazil, and the United States (USA) lead the global production, which is mainly devoted to juice extraction, but Spain is the leading country for exports of fresh entire fruit for direct consumption. Among postharvest losses of fresh fruit, those of pathological origin are especially important because whole export shipments are often rejected by wholesale buyers when they find rotten fruits, even at low proportions. In these cases, the producer is also charged for the transport and handling costs.¹ Postharvest diseases of citrus fruits are typically caused by filamentous fungi and, according to the origin of the infections, these fungal pathogens can be classified as wound or latent pathogens. Wound pathogens infect the fruit through peel injuries inflicted in the field, during harvest, transportation, postharvest handling in the packinghouse, or commercialization. They affect citrus worldwide and are the most economically important in citrus production areas with a Mediterranean-type climate, such as Spain or California. Major species are Penicillium digitatum (Pers.:Fr.) Sacc. and Penicillium italicum Wehmer, which cause citrus green and blue molds, respectively.² Latent pathogens infect flowers or young fruit in the grove, but only develop after harvest. They are particularly important in production areas with abundant summer rainfall, such as Brazil or Florida, and cause postharvest diseases such as stem-end rots, anthracnose, brown rot, black rot, and gray mold.

The management of postharvest diseases in citrus packinghouse has been for many years and still today primarily based on the application of conventional chemical fungicides, such as imazalil (IMZ), sodium ortho-phenyl phenate (SOPP), Pathogens react vigorously to their environment; even the slightest variations in the environment may greatly impact the pathogen and result in minute to massive adaptations in its population.¹ The extent and rate of such adaptations brought about in a pathogen shapes its morphological and physiological characteristics, and also changes its behavior towards the host population. Alternaria species have been extensively studied for their differential pathogenicity/virulence on different host species. Variability in virulence towards host species have been reported in isolates of A. brassicae,^2-9 A. brassicicola,^3,10,11 A. alternata¹² and A. solani.^4,13,14 The availability of pathotypes with varying degrees of pathogenicity towards different Brassica species and sub-species acts as an important aspect in identification, breeding and exploitation of durable resistance genotypes.

Induction of resistance in otherwise susceptible host plants, without changing their basic genetic make-up, through use of biotic as well as abiotic agents has been studied by a few scientists.^15,16 The resistance/ defense related genes in the vulnerable plants can be activated by inoculating the plant either by an avirulent form of the pathogen or by limited inoculation with the pathogen.¹⁷ Infecting avirulent pathogen triggers natural defense responses in the plant through the release of the elicitors which then result in the expression of novel anti-pathogenic proteins.

In order to harness the benefits of induced host resistance and build up a stable, long term resistance mechanism in the host plant against the pathogen, there is a need to identify the pathogen and understand its behavior under diversified conditions. However, only a few studies⁷ have been undertaken to evaluate the pathological diversity of the Alternaria isolates and its characterization for its applicability in India.

Raising brassica plants

Six varieties of oilseed Brassica - B. juncea (4 varieties - Varuna, Rohini, Kranti and PHR-01) and B. rapa (2 varieties - YSH-401 and Pusa gold) were selected for evaluating variations in virulence/aggressiveness of different isolates. The seeds of the selected varieties were sown in agropeat: sterile soil mix and plants were raised under natural conditions in field during Rabi Season and controlled conditions at 22 ± 2˚C with 16 hr light/8 hr dark photoperiod and a light intensity of 12klx.

Isolation and purification of fungal cultures

Samples of Alternaria blight infected leaves from various Brassica species were collected/procured from Northern and North-western regions of India. The fungal cultures were isolated and purified on PDA (potato dextrose agar) medium under continuous diffused light conditions, at a temperature of 20 ± 2˚C for 15 ± 2 days. The purified Alternaria isolates were observed microscopically under 40× optical microscope and identified at species level as per the available monograph.¹⁸

Pathogenicity assay

The differential virulence/aggressiveness of the isolates were determined using detached leaf according to the method of Vishwanath & Kolte.¹⁹ The maintained isolates were used to prepare individual spore suspensions (1.5 × 104 spores per ml). Third/fourth fully expanded leaf from base of 30-day old plants of each variety were detached, inoculated with 10 μl of individual spore suspensions and incubated for 7 days at 22 ± 2˚C and 12 hour photoperiod in moist chambers.

The response of different isolates on the selected hosts was assessed on the basis of lesion number, size and colour; presence of ring/dots in the lesion; chlorotic zone (yellow halo) and latent period of infection, in terms of Disease Index (DI) on a scale of 0-9 (Table 1). The isolates were subsequently characterized as highly aggressive, aggressive or non-aggressive on the host differentials and analyzed statistically for significant variations, if any. On the basis of non-aggressiveness shown by the isolates, three varieties of Brassica - B. juncea (2 varieties - Rohini and PHR-01), and B. rapa (1 variety - YSH-401) were selected for further evaluations.

Evaluation for induction of systemic acquired resistance

Assay for evaluation of systemic resistance induced by attenuated virulence was carried out using detached leaf method. A separate assay was carried out for each isolate showing non-aggressive behavior on a specific Brassica variety. The detached leaves from base of 30-day old plant of the respective Brassica variety were inoculated with 10 μl of four different suspensions: sterile distil water as control (C); spore suspensions of non-aggressive isolate; highly aggressive isolate; non-aggressive isolate (on 1st day of inoculation) followed by spore suspension of highly aggressive isolate (on 3rd day of inoculation). The inoculated leaves were incubated for 10 days at 22 ± 2˚C and 12 hr light/12 hr dark photoperiod in moist chambers. Host response to pathogen’s aggressiveness and disease score was assessed as earlier and data was analyzed statistically for induction of systemic resistance.

Pathological diversity

A wide variation was exhibited by A. brassicae, A. brassicicola and A. alternata isolates from different regions on the selected host differentials. The disease reaction varied with necrotic lesions of 0.4-7.5mm, mostly having brown to black colour. A few isolates also formed grey to dark grey coloured lesions. The isolates in general caused moderate to severe chlorosis on B. rapa varieties (YSH-401 and Pusa gold) as compared to the B. juncea varieties (Varuna, Rohini, Kranti and PHR-01), on which the chlorosis was either absent or was very slight.

On a scale of 0-9, disease severity in A. brassicae isolates varied between 2.22 ± 1.17 to 7.32 ± 0.67. A. brassicae isolates showed maximum mean disease index of 5.13 ± 0.38 on B. rapa var. Pusa gold, followed by B. juncea var. Kranti, B. rapa var. YSH-401, B. juncea var. Rohini, Varuna and minimum value of 3.35 ± 0.47 on B. juncea var. PHR-01 (Figure 1 & Table 2). Similarly, A. brassicicola isolates varied in aggressiveness from 2.38 ± 0.92 to 5.89 ± 0.89. A. brassicicola isolates showed highest disease index of 5.05 ± 0.51 on B. juncea var. Kranti, followed by B. rapa var. YSH-401, Pusa gold, B. juncea var. PHR-01, Varuna and a minimum value of 3.51 ± 0.51 on B. juncea var. Rohini (Table 3). Among the three A. alternata isolates, disease severity varied from 2.49 ± 0.97 to 5.87 ± 0.91. A. alternata isolates showed maximum disease index of 6.0 ± 2.1 on B. rapa var. Pusa gold followed by YSH-401, B. juncea var. Kranti, PHR-01 Rohini and a minimum value of 1.87 ± 1.15 on B. juncea var. Varuna. Overall, among the three species, isolates ABc-P10 (A. brassicae isolated from Pantnagar), ABo-L04 (A. brassicicola isolated from Ludhiana) and ABa-L14 (A. alternata isolated from Ludhiana) were most aggressive on all the tested host differentials. Based on their aggressiveness the isolates were subsequently categorized into four groups; non-aggressive, less aggressive, moderately aggressive and highly aggressive on specific host differentials (Table 4).

Induction of systemic resistance against A. brassicae isolates

Inoculation of spore suspension of non-aggressive A. brassicae isolates prior to inoculation with highly aggressive isolates resulted in the significant reduction in disease severity (at P < 0.05) by five out of the seven tested non-aggressive isolates in respective Brassica varieties (Figure 2). In case of B. juncea var. Rohini, inoculation with a non-aggressive isolate (ABc-P01) prior to highly aggressive isolate (ABc-P10), resulted in 44.5% reduction in disease severity, compared to the inoculation with highly aggressive isolate alone. Similarly, in B. juncea var. PHR-01, non-aggressive isolates (ABc-D06, ABc-P07, ABc-B01 and ABc-H05) reduced the disease severity of highly aggressive isolate ABc-P09 by 7.4, 51.9, 3.7 and 11.1 %, respectively. Isolate ABc-H05 also reduced the disease severity against highly aggressive ABc-P06 in B. rapa var. YSH-401 by 18.5%, thereby inducing systemic resistance. However, in contrast to above, two non-aggressive isolates, ABc-P12 and ABc-H04 acted in an additive manner increasing the disease response in B. juncea var. PHR-01 by 26.3 and 3.7% respectively.

Thorough knowledge of the variability existing amongst the pathogen population and their response is the primary prerequisite for understanding any host-pathogen system. Moreover, it is also highly crucial in the process of breeding for resistance/tolerance against a particular disease. A major lacuna in developing resistance/tolerance against Alternaria blight disease of crucifers is the absence of sufficient documentation of pathogen behavior and its variability under different geographical locations. Identification and classification of Alternaria species as well as their pathotypes has been shown to be greatly influenced by the environmental conditions. In view of this, the present study was carried out with 55 Alternaria isolates procured and purified from eight states of north and north-west regions of India. Our earlier study reports the characterization of these isolates on the basis of their morphological, cultural, biochemical and molecular characteristics. Attempt was also made to correlate the total carbohydrate concentration of the isolates with the pathological response towards host; however, contradictory to the results obtained by Vishwanath & Kolte²⁰ consistent direct proportionality was not observed. Also consistent grouping with respect to all the characters studied could not be done because of the wide diversity obtained.³ Pathological assay of the isolates in this study, on selected host differentials has shown significant variations among the isolates at inter and intraspecific levels. Such a level of extensive variations indicates that the level of genetic variance in host resistance and pathogen virulence can strongly influence the population dynamics and equilibrium of the interacting species. The earlier studies on various host-pathogen systems,²¹ including Alternaria-Brassica, are restricted to single Alternaria species, with limited host differentials.^2,5,6,10

The present study reports the existence of pathological variations among the three most common Alternaria species pathogenic to brassicas viz., A. brassicae, A. brassicicola and A. alternata on host-differentials of B. juncea (four varieties) and B. rapa (two varieties) and also between the isolates of these species collected from different locations. The pathological response of the isolates was also found to vary with respect to different hosts. The isolates have shown varied response on the selected host differentials ranging from high to moderately aggressive to non-aggressive. Overall, A. brassicae and A. brassicicola isolates from Pantnagar have consistently shown high aggressiveness varying from 47-69% and 51-77% respectively. Although B. juncea var. Varuna is generally used as a susceptible check for Alternaria blight and White rust by many researchers, the present study shows that the pathological response varies from moderate to high and that all isolates are not highly aggressive on B. juncea var. Varuna. .

In recent times, drastic variations in the pathogen behavior, increased disease severity and resistance to available fungicides have raised a doubt on the sustainability of chemical methods as sole strategy for disease control in Brassicas. In this changing scenario, biocontrol measures for disease management have gained considerable importance. One such method is generation of induced systemic resistance (ISR) in plants i.e. activation of plant’s natural defense mechanisms by prior inoculation with a non-aggressive pathogen, limited inoculum of the pathogen or its products. Reports have shown the utilization of microbial spore suspensions, culture filtrates and plant extracts resulting in reduction of Alternaria blight disease severity in vegetable and oilseed brassicas.^22-26 The reduction in the disease severity in all such cases is due to the resultant Pathogenesis-Related (PR) proteins released via salicylic acid (SA)/jasmonic acid (JA) pathways activated in response to the pathogen attack. Application of ISR as a disease control strategy demands for the identification of the non-aggressive isolates of the pathogen from the diverse gene pool. In our study, among all the isolates evaluated for their aggressiveness, non-aggressive behavior was observed on different host-differentials by ABc-P01 (on B. juncea var. Rohini), ABc-D06, ABc-P07, ABc-P12, ABc-B01, ABc-H04 (on B. juncea var. PHR-01), and ABc-H05 (on B. juncea var. PHR-01 and B. rapa var. YSH-401). These isolates were further tested for induction of systemic resistance. Out of the 7 non-aggressive isolates tested, five isolates (ABc-P01, ABc-D06, ABc-P07, ABc-B01 and ABc-H05) through the process of attenuated virulence were able to reduce the disease severity of corresponding highly aggressive isolates, thereby inducing systemic resistance in their respective hosts. These results support the findings of Vishwanath et al.⁷ who reported the induction of resistance in PR-15 variety of mustard against virulent A. brassicae isolates A and C using avirulent A. brassicae isolate D.

To conclude, the Alternaria isolates purified from different Brassica species from varied regions in North and North-West India showed differential aggressiveness on selected host-differentials and a few A. brassicae isolates also induced systemic resistance against their highly aggressive counterparts. This ability of the attenuated isolates to protect the oilseed brassica plants from challenge inoculation of the more aggressive isolate indicates that immunization can be effective against the disease like Alternaria blight caused by facultative fungal pathogen which is otherwise more obviously known to be the case for obligate fungal pathogens in the field of host-pathogen interactions. The availability of such pathotypes with varying degrees of pathogenicity/aggressiveness towards different Brassica species and sub-species also acts as an important base in identification, breeding and exploitation of durable resistance genotypes. Protection of oilseed brassica leaf infection by naturally occurring antagonist (less or non-aggressive isolates of the pathogen) results in reduction of lesion size with reduced incubation period consequently resulting in reduced inoculum and further helping in slowing down the epidemic development of the disease which can be a very useful component in integrated disease management strategy of Alternaria blight of rapeseed mustard crop.²⁷ Newer isolates/isolates from other regions of India may be evaluated to identify the isolate capable of inducing systemic resistance in a wider range of B. juncea varieties. Integration of ISR-triggering non-aggressive pathotypes of the plant pathogens along with other disease control strategies would in future lead to an eco-friendly mode of attaining sustainable disease resistance in plants.

We are thankful to Dr (s) Dhiraj Singh and AS Rathi (CCSHAU, Hisar), PD Meena (DRMR, Bharatpur), Ashok Kumar (CSKHPKVV, Kangra), Rajendra Prasad (CSAUA&T, Kanpur), Ranbir Singh (NBPGR, New Delhi), and DK Yadava (IARI, New Delhi), for providing Alternaria infected leaf samples. We sincerely thank Ms. Divya Kilam for her help in manuscript preparation. The financial support provided by Department of Science and Technology to carry out this work is gratefully acknowledged. It is also stated that there is no conflict of interest among the authors.

The author declares no conflict of interest.

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