Molecular Mechanism of Resistance to Alternaria alternata Apple Pathotype in Apple by Alternative Splicing of Transcription Factor MdMYB6-like.

As a fruit tree with great economic value, apple is widely cultivated in China. However, apple leaf spot disease causes significant damage to apple quality and economic value. In our study, we found that MdMYB6-like is a transcription factor without auto-activation activity and with three alternative spliced variants. Among them, MdMYB6-like-ß responded positively to the pathogen infection. Overexpression of MdMYB6-like-ß increased the lignin content of leaves and improved the pathogenic resistance of apple flesh callus. In addition, all three alternative spliced variants of MdMYB6-like could bind to the promoter of MdBGLU H. Therefore, we believe that MdMYB6-like plays an important role in the infection process of the pathogen and lays a solid foundation for breeding disease-resistant cultivars of apple in the future.

[Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province].

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

Alternaria koreana sp. nov., a new pathogen isolated from leaf spot of ovate-leaf Atractylodes in South Korea.

BACKGROUND: A new species within the genus Alternaria was isolated from the leaf spot of Atractylodes ovata in the Mungyeong and Hwabuk-myeon districts of the Gyeongbuk province of Korea. The leaves showed disease symptoms such as circular or irregular leaf spots and brown to dark brown with gray spots at the center. The leaves also showed that concentric rings were surrounded with yellow halos. METHODS AND RESULTS: Phylogenetic analysis was conducted using the sequence dataset of the internal transcribed spacer region and part of the glyceraldehyde-3-phosphate dehydrogenase. The RNA polymerase II second largest subunit, endopolygalacturonase, Alternaria major allergen gene, anonymous gene region, and translation elongation factor 1-alpha genes were used as well. Results showed that present fungal isolates were distinct from other species of the sect. Alternaria. Morphologically, the present isolates also differed from other members of the sect. Alternaria in their production of solitary conidia or conidial chains (two units) and conidial body features. Similarly, it exhibited moderate pathogenicity in the host plant. CONCLUSIONS: This study described and illustrated A. koreana as a new species and the causal agent of the leaf-spot disease on A. ovata in Korea.

Distinct and essential roles of bZIP transcription factors in the stress response and pathogenesis in Alternaria alternata.

The ability to cope with environmental abiotic stress and biotic stress is crucial for the survival of plants and microorganisms, which enable them to occupy multiple niches in the environment. Previous studies have shown that transcription factors play crucial roles in regulating various biological processes including multiple stress tolerance and response in eukaryotes. This work identified multiple critical transcription factor genes, metabolic pathways and gene ontology (GO) terms related to abiotic stress response were broadly activated by analyzing the transcriptome of phytopathogenic fungus Alternaria alternata under metal ions stresses, oxidative stress, salt stresses, and host-pathogen interaction. We investigated the biological functions and regulatory roles of the bZIP transcriptional factor (TF) genes in the phytopathogenic fungus A.alternata by analyzing targeted gene disrupted mutants. Morphological analysis provides evidence that the bZIP transcription factors (Gcn4, MeaB, Atf1, the ER stress regulator Hac1, and the all development altered-1 gene Ada1) are required for morphogenesis as the colony morphology of these gene deletion mutants was significantly different from that of the wild-type. In addition, bZIPs are involved in the resistance to multiple stresses such as oxidative stress (Ada1, Yap1, MetR) and virulence (Hac1, MetR, Yap1, Ada1) at varying degrees. Transcriptome data demonstrated that the inactivation of bZIPs (Hac1, Atf1, Ada1 and Yap1) significantly affected many genes in multiple critical metabolism pathways and gene ontology (GO) terms. Moreover,the ΔHac1 mutants displayed reduced aerial hypha and are hypersensitivity to endoplasmic reticulum disruptors such as tunicamycin and dithiothreitol. Transcriptome analysis showed that inactivation of Hac1 significantly affected the proteasome process and its downstream unfolded protein binding, indicating that Hac1 participates in the endoplasmic reticulum stress response through the conserved unfolded protein response. Taken together, our findings reveal that bZIP transcription factors function as key regulators of fungal morphogenesis, abiotic stress response and pathogenesis, and expand our understanding of how microbial pathogens utilize these genes to deal with environmental stresses and achieve successful infection in the host plant.

Effects of silicon dioxide, zinc oxide and titanium dioxide nanoparticles on Meloidogyne incognita, Alternaria dauci and Rhizoctonia solani disease complex of carrot.

Foliar spray of silicon dioxide (SiO2 NPs), zinc oxide (ZnO NPs) and titanium dioxide (TiO2 NPs) nanoparticles were used for the management of Meloidogyne incognita, Alternaria dauci and Rhizoctonia solani disease complex of carrot. Foliar spray of SiO2 NPs/ZnO NPs or TiO2 NPs increased plant growth attributes, chlorophyll and carotenoid of carrot. Foliar spray of 0.10 mg ml-1 SiO2 NPs caused the highest increase in plant growth, chlorophyll and carotenoid content of leaves followed by spray of 0.10 mg ml-1 ZnO NPs, 0.05 mg ml-1 SiO2 NPs, 0.05 mg ml-1 ZnO NPs, 0.10 mg ml-1 TiO2 NPs and 0.05 mg ml-1 TiO2 NPs. Use of SiO2 NPs caused a higher reduction in root galling, nematode multiplication and disease indices followed by ZnO NPs and TiO2 NPs. Two principal components analysis showed a total of 97.84% overall data variance in plants inoculated with single pathogen and 97.20% in plants inoculated with two or more pathogens. Therefore, foliar spray of SiO2 NPs appears interesting for the management of disease complex of carrot.

Introgression and QTL mapping conferring resistance for Alternaria brassicae in the backcross progeny of Sinapis alba + Brassica juncea somatic hybrids.

KEY MESSAGE: A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC2F3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02 cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae.

Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A.limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg-1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Bioactivites of Penicillium citrinum isolated from a medicinal plant Swertia chirayita.

Endophytes associated with plants have the property to produce active biomolecules with their possible applications in agro-industrial sectors. This study provides a project work on analyzing various activities of fungal endophytes isolated from Swertia chirayita of Sikkim Himalayan region. Among several fungal endophytes screened, isolate UTCRF6 was found most active with the secretion of enzymes protease, cellulase, amylase and chitinase, as well as other metabolites Indoleacetic acid and siderophores. This endophyte was found active in restricting the growth of phyto-pathogens, including strains of Fusarium solani, Colletotrichum gloeosporioides, Alternaria alternata, Pestalotiopsis theae and Sclerotinia sclerotiorum. Morphological and molecular studies of this endophytic fungus showed similarity with Penicillium citrinum.

The Effect of Photoperiod on Necrosis Development, Photosynthetic Efficiency and 'Green Islands' Formation in Brassica juncea Infected with Alternaria brassicicola.

The main goal of growing plants under various photoperiods is to optimize photosynthesis for using the effect of day length that often acts on plants in combination with biotic and/or abiotic stresses. In this study, Brassica juncea plants were grown under four different day-length regimes, namely., 8 h day/16 h night, 12 h day/12 h night, 16 h day/8 h night, and continuous light, and were infected with a necrotrophic fungus Alternaria brassicicola. The development of necroses on B. juncea leaves was strongly influenced by leaf position and day length. The largest necroses were formed on plants grown under a 16 h day/8 h night photoperiod at 72 h post-inoculation (hpi). The implemented day-length regimes had a great impact on leaf morphology in response to A. brassicicola infection. They also influenced the chlorophyll and carotenoid contents and photosynthesis efficiency. Both the 1st (the oldest) and 3rd infected leaves showed significantly higher minimal fluorescence (F0) compared to the control leaves. Significantly lower values of other investigated chlorophyll a fluorescence parameters, e.g., maximum quantum yield of photosystem II (Fv/Fm) and non-photochemical quenching (NPQ), were observed in both infected leaves compared to the control, especially at 72 hpi. The oldest infected leaf, of approximately 30% of the B. juncea plants, grown under long-day and continuous light conditions showed a 'green island' phenotype in the form of a green ring surrounding an area of necrosis at 48 hpi. This phenomenon was also reflected in changes in the chloroplast's ultrastructure and accelerated senescence (yellowing) in the form of expanding chlorosis. Further research should investigate the mechanism and physiological aspects of 'green islands' formation in this pathosystem.

Evaluation of disease resistant and high yielding faba bean germplasm in India.

Faba bean (Vicia faba L.) is one of the earliest domesticated food legumes after chickpea and pea in the world. It is been produced in many countries including China, Ethiopia, Egypt, northern Europe, the Mediterranean region, central Asia, East Asia, Latin America and as a minor crop in India. The crop is affected by many diseases and alternaria leaf blight (Alternaria spp.) is one of the serious threat to faba bean production. Twenty-five lines of faba bean were selected from three international nurseries and were evaluated at ICARDA-FLRP-Amlaha during 2016-2017 and 2017-2018, to identify resistant lines against alternaria blight disease. A wide range of variation to disease reaction was observed among faba bean genotypes. One faba bean line (S2011-134) found tolerant, six genotypes (S2011-116, FLIP15-139, FLIP15-156, FLIP15-159, FLIP15-164-S2 and FLIP15-169) were found moderately tolerant and 16 genotypes were found susceptible to alternaria blight. The faba bean genotypes showed resistance to the disease scoring (0-9) with high yield as compared to the checks, Giza and Gwalior local. The identified sources of resistance can be utilized in faba bean breeding programmes for the development of disease tolerant cultivars with high yield.

Molecular characterization of leaf spot caused by Alternaria alternata on buttonwood (Conocarpus erectus L.) and determination of pathogenicity by a novel disease rating scale.

The buttonwood (Conocarpus erectus L.) is a mangrove shrub belonging to Combretaceae family. It mostly grows on the shorelines of tropical and subtropical regions in the world. It was introduced to Lasbela University of Water, Agriculture & Marine Sciences (LUWMS), Uthal, Baluchistan as an ornamental plant as it grows well under harsh, temperate and saline conditions. During a routine survey, typical leaf spot symptoms were observed on the leaves of buttonwood plants. A disease severity scale for alternaria leaf spot of buttonwood was developed for the first time through this study. Disease severity according to the scale was 38.97%. The microscopic characterizations was accomplished for the identification of Alternaria alternata and Koch's postulates were employed to determine the pathogenicity. For molecular identification, 650 bp internal transcribed spacer (ITS) regions (ITS1, 5.8s and ITS2) were amplified from three representative isolates (LUAWMS1, LUAWMS2 and LUAWMS3) through polymerase chain reaction (PCR). The nucleotide sequences from ITS regions of the isolates were submitted to NCBI with GenBank accession numbers MW585375, MW585376 and MW585377, respectively. The phylogenetic tree of 22 A. alternata isolates was computed and representative isolates exhibited 99.98% genetic similarity with mangroves ecosystem isolates. This study reports the incidence of alternaria leaf spot of buttonwood at LUWMS for the first time. It is suspected that the disease may spread further. Therefore, effective management strategies should be opted to halt the further spread of the disease.

Antifungal Biphenyl Derivatives from Sorbus pohuashanensis Leaves Infected by Alternaria tenuissi and Their Effect against Crop Pathogens.

Eight natural biphenyl-type phytoalexins exhibiting antifungal effect were isolated from the leaves of Sorbus pohuashanensis, which invaded by Alternaria tenuissi, and their growth inhibition rate towards A. tenuissi were 50.3 %, 54.0 %, 66.4 %, 58.8 %, 48.5 %, 51.0 %, 33.3 %, and 37.0 %, respectively. In vivo activity assay verified the protective effect of these natural biphenyls on tobacco leaves. The observation of mycelial morphology revealed that these compounds possessed adverse effects on mycelial growth of A. tenuissi. Subsequently, the most potent active compounds, 3',4',5'-trimethoxy[1,1'-biphenyl]-4-ol (3) and 3,4,4',5-tetramethoxy-1,1'-biphenyl (4), were conducted to the further antifungal evaluation and showed significant activity against the other four crop pathogens, Fusarium graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum, and Exserohilum turcicum. Further, the structure-activity relationships and biosynthesis of these compounds were speculated in this work.

Evaluation of Seven Essential Oils as Seed Treatments against Seedborne Fungal Pathogens of Cucurbita maxima.

Essential oils are gaining interest as environmentally friendly alternatives to synthetic fungicides for management of seedborne pathogens. Here, seven essential oils were initially tested in vivo for disinfection of squash seeds (Cucurbita maxima) naturally contaminated by Stagonosporopsis cucurbitacearum, Alternaria alternata, Fusarium fujikuro, Fusarium solani, Paramyrothecium roridum, Albifimbria verrucaria, Curvularia spicifera, and Rhizopus stolonifer. The seeds were treated with essential oils from Cymbopogon citratus, Lavandula dentata, Lavandula hybrida, Melaleuca alternifolia, Laurus nobilis, and Origanum majorana (#1 and #2). Incidence of S. cucurbitacearum was reduced, representing a range between 67.0% in L. nobilis to 84.4% in O. majorana #2. Treatments at 0.5 mg/mL essential oils did not affect seed germination, although radicles were shorter than controls, except with C. citratus and O. majorana #1 essential oils. Four days after seeding, seedling emergence was 20%, 30%, and 10% for control seeds and seeds treated with C. citratus essential oil (0.5 mg/mL) and fungicides (25 g/L difenoconazole plus 25 g/L fludioxonil). S. cucurbitacearum incidence was reduced by ~40% for plantlets from seeds treated with C. citratus essential oil. These data show the effectiveness of this essential oil to control the transmission of S. cucurbitacearum from seeds to plantlets, and thus define their potential use for seed decontamination in integrated pest management and organic agriculture.

The transcription regulator ACTR controls ACT-toxin biosynthesis and pathogenicity in the tangerine pathotype of Alternaria alternata.

The host-selective ACT toxin is essential for the pathogenesis of the citrus fungal pathogen Alternaria alternata. However, the mechanism of ACT-toxin gene clusters ACT-toxin biosynthesis regulated by is still poorly understood. The biosynthesis of ACT toxin is mainly regulated by multiple ACT toxin genes located in the secondary metabolite gene cluster. In this study, we reported a transcription regulator ACTR contributes ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 in Alternaria alternata. We generated ACTR-disrupted and -silenced mutants in the tangerine pathotype of A. alternata. Phenotype analysis showed that the ACTR mutants displayed a significant loss of ACT toxin production and a decreased virulence on citrus leaves whereas the vegetative growth and sporulation were not affected, indicating an essential role of ACTR in both ACT toxin biosynthesis and pathogenicity. To elucidate the transcription network of ACTR, we performed RNA-Seq experiments on wild-type and ACTR null mutant and identified genes that were differentially expressed between two genotypes. Transcriptome profiling and RT-qPCR analysis demonstrated that the ACT toxin biosynthetic gene ACTS4 is down-regulated in ACTR mutant. We generated ACTS4 knock-down mutant and found that the pathogenicity of ACTS4 mutant was severely impaired. Interestingly, both ACTR and ACTS4 are not involved in the response to different abiotic stresses including oxidative stress, salt stress, cell-wall disrupting regents, and metal ion stress, indicating the function of these two genes is highly specific. In conclusion, our results highlight the important regulatory role of ACTR in ACT toxin biosynthesis through mediating ACT toxin synthesis gene ACTS4 and underline the essential role of in the tangerine pathotype of A. alternata.

Curvularia lunata as new causal pathogen of tomato early blight disease in Egypt.

Tomato plants displaying early blight symptoms were collected from different localities in the provinces of Assiut and Sohag, Egypt. The causal pathogens were isolated on potato dextrose agar plates. Pathogenicity tests with 48 isolates were carried out under greenhouse conditions on tomato cultivar (CV 844). All tested isolates caused symptoms of early blight disease with different degrees. The highest disease severity on tomato plants was found after inoculation with isolate No. 6 followed by isolates No. 20 and No. 31. The most pathogenic isolates were identified by sequence analysis using ITS1 and ITS4 primers. The analysis of the amplified sequences from fungal isolates No. 6, 20 and 31 displayed 99-100% nucleotide identity with Alternaria solani, Curvularia lunata and A. alternata, respectively. To our knowledge, this is the first report of Curvularia lunata as one of the causal pathogens of early blight disease of tomato plants in Egypt.

The effect of a consortium of Penicillium sp. and Bacillus spp. in suppressing banana fungal diseases caused by Fusarium sp. and Alternaria sp.

AIMS: This study sought to utilize indigenous soil micro-organisms to suppress wilt-causing fungal pathogens of the banana. METHODS AND RESULTS: Fungal pathogens were isolated from wilt-affected rhizospheric soil, and potential antagonistic bacterial strains were isolated from healthy rhizospheric soil in the same area from which fungal pathogens were isolated. The antifungal activity of isolated micro-organisms against fungal pathogens was studied both in vitro and in vivo against fungal pathogens. It was found that Fusarium oxysporum and Alternaria sp. were pathogenic, while Penicillium sp., Bacillus velezensis and Bacillus subtilis were antagonistic. Moreover, it was seen that B. velezensis, B. subtilis and Penicillium sp. inhibited the growth of the two fungal pathogens in both in vitro and in vivo experiments. Further investigation indicated that B. velezensis, B. subtilis and Penicillium sp. were able to produce enzymatic antifungal compounds (chitinase and ß-1,3-glucanase). The spray application around rhizome revealed that a combination of Bacillus spp. and Penicillium sp. in greenhouse conditions gave the highest reduction in disease severity by up to 60% to both fungal pathogens among the treatments. CONCLUSIONS: Banana disease is seen to be induced not only by F. oxysporum but also by Alternaria sp. The isolated indigenous micro-organisms can effectively control both the pathogens. The combination of isolated antagonistic micro-organisms has thus demonstrated substantial potential for suppressing banana disease. SIGNIFICANCE AND IMPACT OF THE STUDY: An antagonistic consortium isolated in this study has demonstrated remarkable potential for controlling fungal diseases caused by Fusarium sp. and Alternaria sp. Therefore, the use of indigenous microflora to improve disease suppression of banana plants against soil-borne pathogens is a preferable approach.

Antimicrobial activity of the volatile compound 3,5-dichloro-4-methoxybenzaldehyde, produced by the mushroom Porostereum spadiceum, against plant-pathogenic bacteria and fungi.

AIMS: In this study, volatile compounds released from mycelia of some aromatic mushrooms were investigated for their inhibitory activity against plant-pathogenic bacteria and fungi. METHODS AND RESULTS: A screening revealed that volatile compounds from mycelia of Porostereum spadiceum remarkably inhibited the colony formation of plant-pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum while also inhibiting the conidial germination of plant-pathogenic fungi including Alternaria brassicicola and Colletotrichum orbiculare. The volatile compounds were isolated from the culture filtrate of P. spadiceum, and 3,4-dichloro-4-methoxybenzaldehyde (DCMB) was identified as a major compound. DCMB significantly inhibited bacterial colonization at 10 µg ml-1 and fungal conidial germination at 0·1-1 µg ml-1 as a vapour. CONCLUSIONS: This is the first report on the production of the volatile compound DCMB by P. spadiceum and on the antimicrobial activity of DCMB against plant-pathogenic bacteria and fungi at low concentrations. It may be possible to use the compound as an agent for protecting crops from bacterial and fungal diseases during cultivation and storage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an understanding of antimicrobial activity of the mushroom volatile compound that may be useful as a novel biological control agent for protecting various plant diseases.

Tryptophan-derived metabolites and BAK1 separately contribute to Arabidopsis postinvasive immunity against Alternaria brassicicola.

Nonhost resistance of Arabidopsis thaliana against the hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive resistance and CYP71A12 and CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. We here revealed that CYP71A12, CYP71A13 and PAD3 are critical for Arabidopsis' postinvasive basal resistance toward the necrotrophic Alternaria brassicicola. Consistent with this, gene expression and metabolite analyses suggested that the invasion by A. brassicicola triggered the CYP71A12-dependent production of indole-3-carboxylic acid derivatives and the PAD3 and CYP71A13-dependent production of camalexin. We next addressed the activation of the CYP71A12 and PAD3-dependent postinvasive resistance. We found that bak1-5 mutation significantly reduced postinvasive resistance against A. brassicicola, indicating that pattern recognition contributes to activation of this second defense-layer. However, the bak1-5 mutation had no detectable effects on the Trp-metabolism triggered by the fungal penetration. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (1) CYP71A12 and PAD3-related antifungal metabolism that is not hampered by bak1-5, and (2) a bak1-5 sensitive immune pathway that activates the expression of antimicrobial proteins.