Abstract: Among different Brassica, Indian mustard (Brassica juncea), an important and predominant rabi oilseed crop of rapeseed mustard group in India, occupies 90% area of the total hectarage. Mustard encounters number of foliar diseases, among them Alternaria blight is the most devastating, causing yield loss up to 35-38%. Chemical properties as well as fatty acid profile govern oil superiority. Besides, the disease undesirably distresses seed quality by reducing seed size, seed discoloration and decline in oil content. In relevance, recently microsatellite locus ‘ABS28’ has been identified and characterized in terms of cross-transferability to other related and unrelated taxa. Data compiled authenticate that this monomorphic microsatellite marker is greatly particular for A. brassicicola, as no magnification signal has been observed from other closely related Alternaria species. No false positive or false negative analyses were witnessed with the assays showed the stringency of specific primer set ABS28F and ABS28R, consequently letting precise and sensitive revealing of A. brassicicola from cultures and specific plant samples. The assay was noticed to be sensitive, consistently detecting 0.01 ng of genomic DNA per PCR of the target fungi. Thus, marker allows assessing fungal growth from the initial phases of infection, providing a rapid and applied alternative to existing defined markers to differentiate and identify A. brassicicola from synchronously occurring fungus, A. brassicae and other fungi related to rapeseed mustard. Till date, 14 monomorphic and one polymorphic microsatellite markers have been recognized from the necrotrophic and phytopathogenic fungus A. brassicicola. Although, identified polymorphic marker (ABS1) prerequisites to be established to assist a further study on the genetic assembly of natural A. brassicicola populations and to match the diversity of isolates initiating from distinct host as well as geographical site. This review provides new insights into further exploration of primer set, ABS28F (50-GCTCCCACTCCTTCCGCGC-30) and ABS28R (50-GGAGGTGGAGTTACCGACAA-30) to probably amplify a more specific amplicon of 380 bp and to prove to be useful as an internal diagnostic marker to differentiate and analyze A. brassicicola from A. brassicae and other fungi associated with leaves, stem and siliquae of rapeseed-mustard.
INTRODUCTION
Brassica is the most economically significant genus of Cruciferae family, which includes oil seed crops like mustard (Brassica juncea) and rape (Brassica campestris) as well as different vegetables viz., cauliflower (Brassica oleracea L. var., botrytis L.), cabbage (B. oleracea var., capitata) consumed all over the world. These crops are heavily infected by Alternaria brassicae causing brown to black circular spots on all the above ground parts like leaves, stems, siliquae etc. Among different Brassica, Indian mustard (Brassica juncea) an important and predominant Rabi oilseed crop of rapeseed mustard group in India occupies 90% area of the total hectarage. Chemical properties as well as fatty acid profile govern oil quality1. While studying biochemistry of Brassica juncea varieties, like Sarson T42, Lahi T9, Varuna 59 and Rai Pusa Bold more significant alterations in lipid composition of seed oil of Sarson T42 were observed because of infection by Cuscuta reflexa (an angiosperm parasite), compared to Lahi T9, whereas Varuna 59 and Rai Pusa Bold varieties showed resistance to infection1. Mustard encounters many of foliar diseases, among them Alternaria blight is the most devastating, causing yield loss up to 35-38%2. Besides, the disease adversely affects seed quality by reducing seed size, seed discoloration and reduction in oil content3. The disease also reduces germinability, oil and protein content of seeds. In the absence of resistant donor germplasm of mustard, plant breeding approaches for the development of novel resistant genotypes are still obscure. The most practical way to overcome this situation is to understand inbuilt resistance in the plants. Plants challenged by fungal pathogen exhibit several biochemical defence responses, which include the accumulation of specific metabolites4.
Among several fungal diseases, severe damage of the foliage or seed germination in crucifers occurs due to Alternaria blight caused by Alternaria brassicae (Berk)2,5-7. Black spot of different crucifers have been reported in many countries; Italy8, UK, USA and a number of other European countries9, Canada10,11, Iran12 as well as India13. Cauliflower (Brassica oleracea var., botrytis) and mustard (Brassica juncea) are two major crucifer crops of India, which are fronting severe yield and quality loss in production consequent to dark leaf spot disease caused by Alternaria brassicae (Berk) Sacc14. They can survive in seeds for a number of months at different temperatures as well as relative humidity15,16. Alternaria brassicae reported to cause loss of about 30 and 47% in cauliflower (Brassica oleracea var., botrytis) and Indian mustard (Brassica juncea), respectively17. Morphological features of conidia and conidiophores and sometimes host plant interaction, facilitate the major taxonomic measures for restriction of fungal species18. Though, due to simple and morphological convergence of conidia and facultative parasitism, the taxonomical arrangement of small spored species, as well as host dependent toxin making fungi has been predominantly jumbled results to an ambiguous host range19. Taxonomy of Alternaria on brassicas is principally based on morphology and interaction with host plant of each of the species occurring (A. brassicicola, A. brassicae and A. raphani) that has a different morphology with differentiated shape and size of conidium among Alternaria species20. All profitable cultivars of brassicas are described to have susceptibility to A. brassicae6. Also, there is not a single report on resistance amongst the crucifers against A. brassicae. For developing cultivars resilient to this species, necessitates extensive awareness of pathogen variations existing in crucifers growing in diverse areas. Intensity of Alternaria blight on brassicas differs according to seasons and regions and among crops specified to a region. This may be because of presence of changeability amongst isolates of Alternaria species. The existence of morphological changeability in the isolates of other Alternaria species has been comprehensively reported7,21. Besides, reports on morphological variability in A. brassicae isolates from different regions of India have been well documented22-25.
This review has been aimed to reveal compiled data on the evaluation of A. brassicae isolates collected from crucifer crops based on morphological, cultural, molecular correlation parameters. Besides, it pertains to potential information on the variability in Alternaria brassicae based on the morphological and genetic level and establishment of modern diagnostic techniques for early, precise and speedy detection of Alternaria brassicae and biomarkers generation and validation using different bioinformatics tools.
IDENTITY AND BIOLOGY OF THE PATHOGEN
Berkeley26 recognized the causal fungus on plants of Brassicaceae family as Macrosporium brassicae Berk, renamed later as Alternaria brassicae. It was suggested that the genus could be systematized into six groups depending on fundamental features of conidia length, width and septation with each group specified by a typical species. Wiltshire27 founded the elementary studies on hyphomycetes that reflected to be fundamental to the prevailing concepts of Alternaria, Macrosporium and Stemphylium. Besides, an extensive study pertaining to taxonomy, parasitism and economic importance of this genus was accomplished28. Morphological deviations of Alternaria species were explained followed by division into three sections and projected a strategy for determination of the most common species28-35.
Survival: Contrasting the temperate conditions, existence of the pathogen on host plant debris and/or affected seed in tropical or sub-tropical India has been eliminated36,37. In India, harvesting of oilseed Brassicas generally take place from February-May, while off-season crops are grown in non-traditional areas from May-September, conditionally coupled with harboring of the fungal pathogen by vegetable Brassica crops and alternative hosts (Anagallis arvensis and Convolvulus arvensis) and probably reasoned for transmission of the A. brassicae from one crop-season to another7,36,38.
Host-pathogen interaction: Multi-layered resistance, multi-component-affinity to host dependent toxin dextruxin B (Fig. 1), qualitative and quantifiable expression of phytoalexins, oversensitive response and Ca2+ storage regulates providence of host-pathogen interaction6. Alternaria tolerant Sinapis alba has been found to detoxify dextruxin B39. Attempts to transfer resistance from wild crucifers to cultivated oil seed Brassica were made40. Cellulase enzymes41 and toxins42 are known to be produced by A. brassicae. Though, their particular role in pathogenesis is still obscure. Principally, A. napiformae and A. longipes have been reported on interact with rapeseed-mustard, especially in India43. Signal transduction and expression analysis have been studied in Alternaria brassicae interaction44.
Host-pathogen-environment-interaction: Introductory effort accomplished by researchers specifies effects of temperature, Relative Humidity (RH) and sunshine hours on incidence of the blight on the oil bearing Brassica45-47, describing associations between diverse climate factors and Alternaria blight occurrence through experimental models. However, there was a need to predict precisely the age of crop at first advent of the disease and the hazard that epidemic of the blight will occur on the crop to enable decisions to be taken regarding optimum time for spray of fungicide by farmers and to avoid unnecessary pesticide application. Intensity of Alternaria blight on leaves48 and pods49 was observed rather to be elevated in late seeded crops.
Host resistance: Host resistance is one of the significant ingredients of joined disease management. The regular appearance of this disease with no apparent variability among the released varieties of Indian mustard warrants immediate attention for the control of this disease. Amongst the oil seed Brassica species, namely, B. juncea and B. rapa are more vulnerable to Alternaria blight than B. carinata and B. napus50,51.
| Fig. 1: | Chemical structure of dextruxin B |
A number of sources of tolerance against Alternaria blight have been documented52, including dwarf B. juncea cv53. Referring to mustard, resistance to Alternaria blight has been observed to be connected with leaf enzymes involved in phenolic pathway, e.g., polyphenol oxidase, peroxidase and catalase, higher leaf sugar contents54. Brassica napus, B. carinata and S. alba have comparatively more epicuticular wax than B. rapa and B. juncea and end to be less sensitive to Alternaria blight55. Higher concentration of phenolics in leaves and lower N56 has been connected with resistance to Alternaria blight that is also known to be linked with features like opposition to conidial retention on plant surface like multiple layering of epicuticular wax forming a physical obstruction as a hydrophobic layer to decrease deposition of water-borne inoculum, decline rate of conidia germination and germ tube formation56. Whereas, investigation on machinery of tolerance to Alternaria blight has shown influence of polygene or cluster gene57 with resistance being regulated by nuclear genes of partial dominance, there has also been signal of constituents of resistance being expressively interrelated to each other concerning slow blighting58 and dominance (h) owing to a predominant role in genetic control of time of expression, additive dominance predominant for other disease development factors viz., AUDPC, etc.59. Efforts to breed for resistance for these factors have generated limited success. Since, resistance to Alternaria blight and leaf spots are noticed to be regulated by additive or polygenes, breeding for resistance to these diseases could involve pyramiding of minor genes distressing the disease to provide additive polygene resistance against the disease, introgression of genes from material found resistant, reciprocal recurrent selection or di-allele selective mating57, wide hybridization21, molecular breeding60, transgenic expressing Trichoderma harzianum endo-chitinase gene61 and pollen culture concomitant with sensitivity test to dextruxin B40. Speedy developments in techniques concerning tissue culture, protoplast fusion, embryo rescue, genetic engineering have been observed leading transfer of disease resistance traits across wide cross ability obstructions possible. Transgenic disease resistant plants over-expressing diverse antifungal compounds like Pathogenesis-Related (PR) proteins (chitinase, glucanase, osmotin, etc.) and Ribosome Inhibiting Proteins (RIPs) viz., thionins, defensins and phytoalexins62 to inhibit growth of the pathogen appear less efficacious. Southern analysis of the putative transgenics showed integration of the transgene. Northern and Western analyses evidenced the integrated transgene to be expressed in the transgenics63. Microarray studies have directed that different defense signaling pathways converge to form substantial networks, which control and coordinate regulatory interactions64, inoculating an ecotype of Arabidopsis thaliana with A. brassicicola (an incompatible interaction) or treated A. thaliana with various defense-related signaling molecules, such as Salicylic Acid (SA), Methyl Jasmonate (MJ) and ethylene. Progress in sequencing pathogens or beneficial microflora and the combination of bioinformatics and functional genomics are expected to provide a better understanding of plant-pathogen networks and lead to increase resistance to crop pathogenesis65. In a revolution towards study of functional analysis of genes involved in Alternaria brassicae interaction, use of linear minimal element is stated to provide high throughput targeted gene disruption66. A few expressed sequence tags have been identified in compatible Alternaria brassicae interaction67. Aminobutyric acid has been documented to induce resistance in B. juncea against A. brassicae68.
Biological control: In majority of European countries, organic agriculture has speedily been altered from a farmers’ movement to an institutionalized portion of agricultural policy. In accreditation, agreement with published organic standards is corroborated through annual inspections on farms69. Spray of soil isolates of Trichoderma viride at 45 and 75 days after sowing could administer Alternaria blight of Indian mustard (Brassica juncea) as efficiently as mancozeb70, which have been confirmed later in multi-location trials22. Botanicals e.g., bulb extract of Allium sativum has been described to successfully administer Alternaria blight of Indian mustard71.
Other techniques of Alternaria blight administration: Early seeding of well-stored spotless certified seed after deep ploughing, clean crop growing, well-timed weeding and maintenance of optimum plant population, escaping irrigation at flowering and pod formation phases may aid to manage the disease48. Though the application of species-group specification does not resolve conclusive species limitations within Alternaria, benefits of its use are that it systematizes at the sub-generic level the morphologically diverse assembly of Alternaria species and allows the comprehensive discussion of morphologically related species without becoming excessively restricted due to nomenclatural ambiguity. Besides, the species group concept has delivered an important framework for hypothesis testing in cutting-edge studies on Alternaria phylogeny.
A comprehensive and comparative account of morphological diversity of certain Alternaria species taking place on cucurbitaceous, brassicaceous and solanaceous crops have been described72-74. Nevertheless, others had found that on radish leaves, the disease advanced at a temperature range of 15-25°C and 100% relative humidity for 10-12 h75. Most of the pathogenic species of Alternaria are known to be prolific toxin producers, ultimately facilitating their necrotrophic lifestyle. Alternaria produces toxins, likely to be accountable for these lesions. Understanding the mechanism of action, predominantly, Host Specific Toxins (HSTs) offer a better appreciation of host pathogen connections and resistance phenomena. Two methodologies have been employed to study the mode of action of HSTs. A principal one is the investigation of the molecular level concerning host selectivity and the other at the cellular level. It reasons amongst other deviations in chloroplast and mitochondria. Besides, it causes severe infection on most of the family Brassicaceae viz., Brassica nigra, Brassica campestris Linn, Brassica oleraceae, Raphanus sativus, Iberis amara as a result of which the quality and quantity of crop decreases36.
Symptoms caused due to Alternaria brassicae: Symptoms of the disease have been described by development of spots on stem, leaves as well as siliquae by A. brassicae76, appearing to be generally grey in color rather than black sooty velvety spots created by A. brassicicola. Spots created by A. raphani appear to be different yellow halos around them. Conversely, the symptoms may vary in accordance to the host as well as environment. Progressively, the disease expresses on middle and upper leaves in the form of smaller sized spots, once defoliation of lower leaves occurs. Subsequently, round black conspicuous spots visualize on stem and siliquae both. These specific spots may combine leading to widespread blackening of siliquae concomitant with wilting of the stem with creation of extended lesions. The infection of Alternaria blight on leaves and silique has been documented to considerably decline the photosynthetic area. The stage of infection on silique unfavorably affects the normal seed development, seed weight, color of seed and percent oil content in seed concomitant with the see quality.
The disease also reduces germinability, oil content and protein content of seeds. Variability in the morphological characteristics in A. brassicae isolates of different regions of India have been reported22-24. Some researchers have studied on cultural variability in Alternaria spp. in respect of mycelial growth and sporulation on different temperature, relative humidity, hydrogen ion concentration and light77 and media78. Variability based on morphology, sporulation, growth and other cultural characteristics have also been reported earlier23. Sharma et al.14 evaluated 32 representative Indian geographical isolates of Alternaria brassicae, the causal agent of Alternaria blight of cauliflower (Vegetable) and rapeseed-mustard (Oilseed). All the isolates showed high level of variability in vitro in respect of conidial length, width and number of septa. Conidia of Uttar Pradesh (India) isolate (CaAbU4) were smallest in size with lowest number of septa. Cluster analysis of data on cultural variability among 32 A. brassicae isolates found a close relationship among isolates of both origins viz., from cauliflower and mustard. Isolates from Uttar Pradesh, Delhi, Haryana and West Bengal were found to be more similar to each other whereas the Rajasthan isolates along with Tamil Nadu and Kerala isolates were distantly related to others. All the isolates were pathogenic in nature but not directly related to the cultural and the morphological characteristics. These isolates were further molecularly characterized by using internal transcribed spacer region where all the isolates were found 56% similar to each other and 99% similar to the A. brassicae isolates present in NCBI database14.
Deviation among the Indian isolates of Alternaria brassicae has also been observed22. The morphological and cultural characteristics are not sufficient for pathogen characterization as these are easily influenced by environmental factors and unable to emphasize genetic information of isolates79. Polymerase Chain Reaction (PCR) has been widely assessed in the field of mycology for diverse genetic analysis, specific detection, phylogenetic studies for identification and taxonomical demarcation. These molecular studies require a number of laborious steps, including preparation of a DNA template by extraction of DNA from fungi80. The omission of the DNA extraction procedure, significantly decreases time and cost and also avoid the risk of contamination during the DNA extraction process. Thus, PCR assay has been employed for molecular diagnostic of any pathogens81.
Variations at DNA level among A. brassicae (Berk.) Sacc., A. brassicicola (Schwein) Wiltshire, A. raphani Groves and Skolko and A. alternata (Fr.) Keissl have been established82,83 by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD). Insight has recently been gained into genes being expressed during Alternaria infection of Brassica84. The researcher used suppression subtractive hybridization between RNA isolated from spores of A. brassicicola incubated in water and on the leaf surface of an ecotype of A. thaliana followed by cloning and sequencing of cDNA clones that were differentially expressed.
In recent years, various molecular tools have been brought in practice for delimitation of fungal taxa, which were designated earlier, based on morphological and host range criteria. The PCR is one of the most popular techniques. It opens a new age in genetic analysis on a molecular level85. Primer is a significant component of PCR and its design is basically important in PCR based detection approaches. The general criteria for primers are quite simple86 but it is difficult to choose primers for a given template sequence. Therefore, the computational aid on primer design is a critical issue in bioinformatics86. There are several web-based services or standalone software provided to the public for primer design, such as PRIDE, PRIMER MASTER, PRIMO, primer blast and prime. However, most of them only take a single sequence query. Besides, the calculation of the primer annealing condition is often simplified in a short equation of melting temperature™, regardless of the sequence content of the primer itself.
Methods for the localization of molecular markers such as RFLP, RAPD, Amplified Fragment Length Polymorphism (AFLP) and Sequence Characterized Amplified Regions (SCAR) have been used extensively to localization genes of interest and in molecular-assisted plant breeding87. Comparison between the rDNA sequence of Alternaria linicola strains88 or Alternaria species pathogenic to crucifers82 revealed variation in the ITS sequence both in base composition and in length, while there is a high degree of homology among species and strains of Alternaria. By analysis of the ITS region in common air borne allergenic fungi, including A. alternata, A. brassicicola, A. brassicae, A. infectoria, A. tennuissima and A. rapahni89 concluded that the inter-species similarity in ITS1 and ITS2 regions will make the design of probes to distinguish between these genera comparatively straight forward. A. alternata could be distinguished from other genera present in tomato products by PCR but the specificity of the primers was not verified against other Alternaria species and closely associated genera like Stemphylium and Ulocladium90. The RAPD analysis has also been brought in practice to study the genetic variation in the genus Alternaria33 among A. solani and A. alternata isolates from potato as well as tomato91.
Primers called as Universal Rice Primers (URP) were developed from the repetitive sequences derived from the rice genome and universally have been employed in PCR based genomic DNA fingerprinting of various organisms covering plants, animals and microorganisms92. The genotype analysis of Alternaria brassicicola was accomplished by microsatellite neutral primers, developed by Avenot et al.93 including 16 pairs of specific primers.
Development of diagnostic biomarker for the rapid documentation of Alternaria brassicae: It is very difficult to administer the disease, due to no proven source of resistance described till date in any of the hosts94. The yield loss due to this pathogen is about 50% in the entire mustard growing area13. One of the noteworthy aspects of biology of an organism is the morphological and physiological features of an individual within a species, which are not fixed. This appears to be factual with fungi too, though it is not is not regular feature in asexually created individuals of the progeny. Changeability studies are imperative to verify the variations happening in populations and individuals as inconsistency in morphological and physiological traits highlight the existence of different pathotypes. Alternaria blight severity on oilseed Brassicas differ season to season, region to region and individual crop to crop in India50. This might be due to the presence of variability among geographically similar isolates of A. brassicae. The variability is a well-known phenomenon in genus Alternaria and may be observed as changes in spore shape and size, growth and sporulation, pathogenicity, etc. Diversity seems even in single spore isolates. Many reports on the existence of changeability among different Alternaria species concerning different hosts have been documented by earlier researchers35,95-97 as also within A. brassicae species23. Recently, Meena et al.98 studied the assertiveness, diversity and distribution of A. brassicae isolates infecting oilseed Brassica in India. Deviation in pathogen populations can usually be identified with the aid of certain approaches like morphological, cultural, pathogenic and molecular specificity.
In a recent report, Goyal et al.25 reported variation in conidial morphology, mycelial growth, sporulation of 13 isolates of A. brassicae collected from different geographical zones were dependent on temperature and geographical origin. Similarly, variability in the morphological features in A. brassicae isolates of different regions of India has been reported22-24. Some researchers have worked on cultural variability in Alternaria spp. in respect of mycelial growth and sporulation23,77,78. Cauliflower and mustard are winter crops and affected by A. brassicae. Along these notions, another very recent study has been noticed to understand the variability among the isolates infecting both the crops grown in different parts of Uttar Pradesh, India based on morphology, cultural and molecular level99. Significant variation in growth, sporulation and conidial morphology of A. brassicae isolates were found on media irrespective of crop and geographical sites.
Information of variability within pathogen inhabitants has pronounced worth in accepting the disease epidemics, its prediction and determining a particular disease management approach99,100. Alternaria brassicae is an economically significant pathogen of Brassica crops in many parts of the world. Variation in Alternaria brassicae has been characterized because of growth, cultural physiognomies, sporulation, morphology and reaction of the commercially cultivated Brassica species. Nevertheless, these techniques are time consuming and highly influenced by environment99.
The DNA markers have been established as a powerful tool to study taxonomy and molecular genetics of a number of organisms. A molecular biology technique such as RFLP has been considered to be worthwhile in detecting variation in fungal species100, though it RFLP is very expensive, laborious and time consuming. Development of RAPD analysis, based on PCR has attracted considerable attention because of its ease of use and rapid generation of reliable and reproducible DNA fingerprints. This technique has already been employed for detecting variation in various pathogenic fungi. The RAPD permits rapid assessment of genetic changeability and has been brought in practice to study inter- and intra-specific changeability among the isolates of numerous fungal species. Certain reports have been documented concerning the genetic distinction intra- and inter-Alternaria spp., through RAPD molecular markers95,97,101,102.
The genetic linking among the isolates of A. brassicae has been effectively analyzed by random primers to create reproducible polymorphisms99. All amplified products revealed banding designs that directed the presence of genetic multiplicity. The RAPD has been employed earlier to course inter- and intra-specific genetic distinction in fourteen isolates of Alternaria brassicae, which were gathered from different geographical regions of the world102. Further, the data obtained by Singh99 showed genetic divergence among the 21 isolates of A. brassicae isolated from mustard. The genetic polymorphism within an Alternaria species has been positively studied employing RAPD molecular markers by many researchers101,102. Genetic polymorphism among A. brassicae isolates from different geographical regions around the world was studied and low intra-regional variation among Indian and Canadian isolates of A. brassicae with 75% resemblance among them was documented102. This genetic changeability within an Alternaria species might be due to the existence of heterokaryosis, mutation, somatic hybridization, host selection, extensive dispersal or of a cryptic sexual phase.
Genetic changeability was observed among 21 isolates of A. brassicae isolated from different cultivars of mustard growing in different parts of Uttar Pradesh, India. This could be the possible reason behind extreme and different disease reactions of genotypes at maximum locations. Several reports are accessible on the genetic variation within and between Alternaria species by RAPD molecular marker95,97,99102. Correspondingly, RAPD has been employed earlier to investigate inter- and intra-specific variation in 32 Alternaria brassiae species, which were isolated from cauliflower (Brassica oleracea L. var., botrytis) and mustard (Brassica juncea L. var., Czern) growing areas in India14.
Expressed Sequence Tags (ESTs) are the source of microsatellites, which can be employed to develop molecular markers for studies on the population genetics of A. brassicicola and closely associated A. brassicae, accountable for leaf spot of rapeseed-mustard. Usually, success rate of EST-SSR primers (percentage of SSR primers producing discrete amplification products) were observed to range from 50-100% between species within plants’ genera103,104 and cross species transmission of microsatellites within genera in fungi was more than 30%105. Forty-five percent amplification of EST derived microsatellites loci in A. brassicicola population obtained in the present investigation is in corroboration to the findings of Benichou et al.106. In cross-species amplification assay, 45% microsatellites loci of A. dauci showed amplification signal for A. bataticola, A. solani and A. zinnia. Similarly, Dracatos et al.107 tested 55 primer pairs designed from the Puccinia coronata f.sp., lolii ESTs to amplify the DNA from various fungal species (Puccinia coronata f.sp., avenae, Puccinia striiformis f.sp., tritici, Neotyphodium lolii, Blumeria graminis, Aspergillus nidulans and Penicillium marneffei) and found that 22-53% amplified. This recommends that microsatellites developed from EST sequences are extremely transferable to other associated species. The more closely connected the organisms, the higher the rate of transferability because of more closely related species sharing more homology in microsatellite loci. Another probability for high rate of success in the amplification of EST-SSRs may be the consequence of numerous factors, such as the sequences from which the primers were derived, the adequate criteria used for primer design and use of the species of same genus for the design and amplification of the primer set99,108. A fascinating aspect of present study is the presence of high percentage of monomorphic SSR loci in A. brassicicola population. This may be due to environmental and survival based adaptations of fungus, which vary from habitat to small niche. There could be more explanations for the presence of monomorphic microsattelite in A. brassicicola, (i) It reasons leaf spot on mustard and cauliflower, both have the same cropping period and environmental conditions and appears very late in season with low intensity, probably leading to less variability in population, (ii) The number of isolates assessed was small and the genetic analysis was of low resolution, (iii) There were repeated bottlenecks and/or single founder population/pathogen mating systems, causing in homogenizing effect, (iv) Microsatellite recognized may be located in the conserved region of the A. brassicicola genome and (v) The incidence of A. brassicicola is less common over A. brassicae in India. All these arguments are reasonable, as evidenced by highly clonal nature with no known teleomorph of A. brassicicola109. Configurations of cross-species SSR amplification in fungi are beginning to emerge, though there are still few studies, which systematically explore microsatellite transferability out there closely related genera92,105,110. Five EST-SSR markers established from A. brassicicola, amplified A. brassicae isolates and showed very low levels of polymorphism99. Analysis of limited number of A. brassicae isolates might be one of the probable explanations for low level of transferability of microsatellites99. Additionally, the wide distribution of A. brassiscae in India14,98-100 over all mustard growing areas may lead to wide changeability in the population that may be possible reason for low level of transferability of microsatellites from A. brassiscicola. It is worth mentioning here that a small number of markers (4 of 25) have also been designated as transferable from linked Uredinales species to Hemileia vastatrix111. The primary results obtained in the recent study accomplished by Singh et al.100 come to an agreement with previous reports, describing a smaller fraction of cross-species transfer of microsatellites within fungal genera112. Nevertheless, there might be a high probability of transferability of A. brassicicola derived markers to A. brassicae, which necessities further investigation and statistical validation using large set of EST-derived SSRs. The results also established wide species transferability of developed EST primers and demonstrated that they may represent a set of well-conserved loci across the species. This may be consequent to the transfer of lineage specific genomic regions in fungi113,114. It has been noticed from the study that the distribution of microsatellite in the A. brasssicola genome is not random. Tri-nucleotide repeats (AAC, GAA and TAC) have been found to be common feature in EST derived microsatellites. High occurrence of these repeats in coding regions could be as a result of mutation and selection procedure for specific amino acids. The abundance of tri-nucleotide repeats EST-SSR is likely due to suppression of other kind of repeats in the coding region that reduces the frame-shift mutations in the coding regions115. Additionally, there is a possibility that these tri-nucleotides in the coding region are translated into amino acid repeats (histidine, glutamic acid, threonine, serine, arginine, asparagine, glycine, etc.), which possibly contribute to the biological function of protein100,116,117. Specific and speedy detection is very significant to monitor and quantify the occurrence of plant pathogens for efficient management of plant diseases. Presently, most of the assays used in A. brassisicola disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting118.
Recently, microsatellite locus ‘ABS28’ was recognized and characterized in terms of cross-transferability to other associated and dissimilar taxa. Data confirm that this monomorphic microsatellite marker was highly specific for A. brassicicola because no amplification signal was observed from other closely connected Alternaria species. No false positive or false negative results were observed with the assays revealed the stringency of specific primer set ABS28F and ABS28R, thus allowing specific and sensitive detection of A. brassicicola from cultures and plant samples. The assay was sensitive, reliably detecting 0.01 ng of genomic DNA per PCR of the target fungi. Consequently, the marker allows assessing fungal development from the initial stages of infection and eventually provides a fast and practical alternate to currently designated markers to differentiate and diagnose A. brassicicola from synchronously occurring fungus, A. brassicae and other fungi connected with rapeseed mustard99,100,110,119.
CONCLUSION
Fourteen monomorphic plus one polymorphic microsatellite markers have been recognized from the necrotrophic phytopathogenic fungus A. brassicicola. Although the identified polymorphic marker (ABS1) needs to be developed to enable a further study of the genetic conformation of natural A. brassicicola populations and to match the diversity of isolates originating from different host as well as geographical location. Furthermore, primer set, ABS28F (50-GCTCCCACTCCTTCCGCGC-30) and ABS28R (50-GGAGGTGGAGTTACCGACAA-30), intensified a specific amplicon of 380 bp and would be advantageous as an internal analytical marker to differentiate and diagnose A. brassicicola from A. brassicae and other fungi linked with leaves, stem and siliquae of rapeseed-mustard.
SIGNIFICANCE STATEMENTS
| • | The study provides complete knowledge of Alternaria blight on Brassica juncea |
| • | This review provide comparison of Alternaria brassicicola and Alternaria brassicae and their effect on Brassica juncea |
| • | It also provides the most updated information on morphological, biochemical and molecular, studies carried out on Alternaria blight |
ACKNOWLEDGMENT
This study could be possible as a consequence of collaboration between Department of Biotechnology, IFTM University, Moradabad and Department of Microbiology, NBAIM, Mau in Uttar Pradesh, India. Authors are grateful to Indian Council of Agricultural Research, New Delhi for providing senior research fellowship to Ruchi Singh for successfully accomplishing this work, a part of her Ph.D. program.