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Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

China has the largest area of kiwifruit production in the world. Pathogens associated with root diseases of kiwi trees have not been investigated extensively. In this research, three Phytophthora species, Phytophthora cactorum, Phytophthora cinnamomi, and Phytophthora lateralis, which are pathogenic to kiwi trees in the main planting areas of China, were studied. The population densities of these species in 128 soil samples from 32 kiwi orchards in 2017 and 2018 were measured using multiplex real-time quantitative PCR based on the ras-related protein gene Ypt1. P. cactorum was the most widely distributed of the three species in orchards of the Zhouzhi and Meixian prefectures. We used redundancy analysis to examine soil factors in the kiwi orchards to understand their effects on the population densities of the Phytophthora species. The redundancy analysis indicated that soil temperature and pH were significantly correlated with the abundance of P. cactorum and P. cinnamomi. In addition, two loop-mediated isothermal amplification detection systems for P. cactorum were developed based on the tigA gene. The color-change detection system proved to be accurate, sensitive, and faster than quantitative PCR. The results of this study, along with the loop-mediated isothermal amplification detection systems, will be of great use in the control of Phytophthora diseases for the production of kiwifruits in China.

The Colletotrichum gloeosporioides species complex contains plant pathogens linked to Anthracnose diseases afflicting various crops. In this study, we designed a loop-mediated isothermal amplification assay (LAMP) primer set based on calmodulin gene coding region sequences from taxonomically authorized isolates of species from this complex to rapidly detect the presence of fungi associated with Anthracnose diseases. This test can be employed at any point between cultivation and sale. Moreover, we examined the specificity and detectable range of this primer set using isolates selected from species of the genus Colletotrichum. This test was able to specifically detect members of the C. gloeosporioides species complex, including C. gloeosporioides, C. aotearoa, C. fructicola, C. horii, C. kahawae, C. musae, C. siamense, C. theobromicola, and C. tropicale. Key Words: Anthracnose, diagnosis, phylogeny, plant disease

Streptomyces sp. strain MBCN152-1, isolated from cabbage, has potential as a biocontrol agent for Alternaria brassicicola on cabbage seedlings. The present study examined its mode of action. Light microscopy showed that appressorium formation by A. brassicicola was significantly suppressed on cabbage seedlings bacterized with MBCN152-1. Furthermore, scanning electron microscopy revealed that the mycelia of MBCN152-1, which were epiphytically growing on the cotyledon leaves of cabbage seedlings, intensively coiled around the germinating conidia of A. brassicicola. In vitro co-culture experiments demonstrated that MBCN152-1 is an aggressive mycoparasite of A. brassicicola, but not of A. brassicae or Colletotrichum higginsianum. Biocontrol experiments indicated that MBCN152-1 did not control diseases caused by A. brassicae or C. higginsianum. These results suggest that mycoparasitism is the primary mode of action for MBCN152-1. This is the first study to clearly demonstrate the significance of mycoparasitism in the biocontrol efficacy of endophytic Streptomyces.

Pythium intermedium plays a vital role in the carbon cycle of cool-temperate forests and is widely distributed in Japan's forest soils. In this study, we performed a phylogenetic analysis of the P. intermedium species complex using DNA sequences from multiple loci. The study included 35 isolates from cool-temperate forest soils, seven known P. intermedium isolates, and six known Pythium attrantheridium isolates. We also performed morphological observations and mating tests. Our results showed that all the isolates formed one large clade but were divided into three subclades. Furthermore, we observed many mating reactions between isolates from different subclades, including between P. attrantheridium and P. intermedium. Therefore, we suggest that P. intermedium, P. attrantheridium, and another phylogenetic species belong to one species complex. This is the first report of a species complex within P. intermedium and will be helpful in understanding the evolution of Pythium species in natural ecosystems.

Phytophthora is an oomycete genus with worldwide distribution, and many of its species cause destructive diseases. In Japan, Phytophthora species are listed as quarantine organisms with the exception of Phytophthora nicotianae. For effective quarantine control, we designed a Phytophthora genus-specific loop-mediated isothermal amplification (LAMP) primer set and a P. nicotianae species-specific quenching probe (QProbe) to establish a simultaneous LAMP-based detection method. We confirmed the specificity of the genus-specific primers, and all 161 taxa were detected. No other species in the closely related genera Pythium and Phytopythium gave positive results with the exception of two species, Phytopythium delawarense and Phytopythium fagopyri. These two species gave inconsistent results. We used annealing curve analysis with the QProbe to demonstrate that P. nicotianae could be distinguished from other species. DNA from inoculated and naturally infected plants was extracted using a time-saving extraction kit and subjected to the simultaneous detection method. We confirmed that all Phytophthora DNAs in the plant samples were detected, and P. nicotianae was specifically identified. This simultaneous detection method will make quarantine inspections faster and easier.

A stem rot disease appeared on hydrangea plants in Tochigi Prefecture, Japan. Four Phytophthora-like isolates were obtained, and a representative isolate was selected for analysis. Hydrangea plants inoculated with the isolate developed similar symptoms. Molecular phylogenetic analyses using the rDNA ITS region and cox1, EF1-α, and β-tubulin genes indicated that the isolate was Phytophthora hedraiandra. The sporangia were papillate, laterally sessile, and caducous. Antheridia were paragynous or amphygynous, and laterally sessile, and the oospores were aplerotic. Based on these characteristics, the isolate was identified as P. hedraiandra. We suggest naming this hydrangea disease “Ajisai kukieki byo”.

China has the largest area of kiwifruit production in the world. Pathogens associated with root diseases of kiwi trees have not been investigated extensively. In this research, three

In Japan, Phytophthoracolocasiae causes leaf blight of taro, resulting in huge losses. To effectively diagnose this pathogen in the field, we developed rapid and reliable methods based on loop-mediated isothermal amplification (LAMP) using LAMP primers designed from the Ypt1 gene that are highly specific with a sensitivity of 100 fg. Among these methods, we concluded that the plant-LAMP method with a heat treatment is simple and dependable for detecting P.Colocasiae in taro cultivation fields and that the plant culture-LAMP is a simple and reliable for identifying P.Colocasiae that has been cultured on media for extended periods.

Hydrogen peroxide (H(2)O(2)) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H(2)O(2) in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H(2)O(2) application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H(2)O(2) but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H(2)O(2) response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H(2)O(2) response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Phytophthora palmivora infects hundreds of plant species around the world. Determining the genetic diversity among isolates is necessary to establish more effective control strategies, manage resistance and understand epidemiology, long-term evolution of the pathogen, host–pathogen coevolution, and evolutionary processes. Here genetic diversity among 81 isolates of P. palmivora from various host plants and geographical regions in Indonesia and Japan was evaluated using rep-PCR (BOX, ERIC, REP and M13) and microsatellite markers. With both methods, the isolates clustered into six groups, which corresponded more to geographic regions rather than host plants or mating types, although the respective members of each group were not always consistent between the two methods. Isolates from Indonesia, Japan and other countries clearly clustered into separate groups in the rep-PCR analysis. The rep-PCR consistently grouped the isolates from Sumatera Island with some isolates from Sulawesi, but microsatellite markers grouped them with those from the Java islands. A typical A1-mating type from one region grouped in one cluster using both methods. Both DNA fingerprinting markers also grouped the isolates from Nusa Tenggara and Papua islands with those from Japan. Genetic variation was recognized within and among populations of P. palmivora based on geographical areas, host plants and mating types. Possible explanations for the results and suggested strategies for disease management are discussed.

In Japan, Phytophthora colocasiae causes the leaf blight of taro, which has resulted in huge losses since 2015. To investigate the causes of disease persistence and expansion, it is important to clarify the basic properties of this pathogen. We collected in total 317 P. colocasiae isolates from 99 fields in 7 prefectures during 2014 to 2020. The mating-type of each isolate was examined, and two or more isolates which were collected in single fields or taro leaves were selected to analyze the mating-type complexity. We found five kinds of mating types were identified: heterothallic A1 and A2, self-fertile (SF) A1, A2 and A1/A2 types, and a complex and diverse distribution of mating-types was present in one field or leaf. In addition, the stability of each mating-type was analyzed by single hyphae, zoospore and zoosporangium. The results suggested that the SF isolates were shown to be genetically unstable, while heterothallic isolates had a stable property. In the pathogenicity test of different mating-type isolates, heterothallic A1 isolates were less pathogenic than heterothallic A2 and SF isolates. However, there was no relationship between the pathogenicity and the growth rate on culture medium.

Afandi A, Subandiyah S, Wibowo A, Hieno A, Afandi, Loekito S, Suga H, Kageyama K. 2021. Population genetics analysis of Phytophthora nicotianae associated with heart rot in pineapple revealed gene flow between populations. Biodiversitas 22: 3342-3348. Phytophthora nicotianae has caused heart rot in pineapple. This study identified the population genetic diversity of P. nicotianae, collecting 90 isolates of P. nicotianae from the pineapple plantation sites in three provinces of Indonesia and, as comparisons, seven isolates from non-pineapple sites. Six polymorphic microsatellite markers amplified the non-coding region was used to characterize the population diversity. The phylogenetic tree constructed by MEGA revealed three major clades; the first and second clades were dominated by the isolates from pineapple plantation sites, and the third clade contained isolates from the non-pineapple sites. The allelic pattern analysis using Genalex software revealed the local alleles specific to Lampung and Blitar populations. The AMOVA of microsatellite genotypes data showed that the isolates had a low diversity among the population (6%) but high diversity within individual. Conclusively, P. nicotianae population associated with heart rot in pineapple showed geneflow between populations.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120‍ ‍min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120‍ ‍min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.

In Kagawa Prefecture, Japan, the pathogens Phytophthora pseudolactucae, Pythium irregulare, Pythium uncinulatum, and Pythium spinosum have caused huge losses in lettuce production. We used loop-mediated isothermal amplification (LAMP) to analyze soil and plants in lettuce fields for the presence of these four pathogens. To develop an effective on-site detection method, we contrasted the Plant-LAMP and Plant Culture-LAMP procedures for plant samples, and five soil DNA extraction methods for soil samples. Plant-LAMP and a Soil DNA Isolation kit were selected to analyze three fields for the pathogen species present, infected sites, and level of soil contamination. We found that the same wilting symptoms could be caused by Phytophthora or Pythium, or a mixture of species from both genera. Ph. pseudolactucae infects the pith of the lettuce in aboveground parts, whereas Pythium spp. mainly infect roots. Ph. pseudolactucae and Py. uncinulatum caused disease more frequently than the other two pathogens. Furthermore, not all of the pathogens existed in the soil near infected lettuce plants. Therefore, the LAMP method can be used to diagnose pathogenic oomycetes in the field, and will be useful in the development of control strategies in lettuce production.

Phytophthora nicotianae is an important soil-borne pathogen in tropical, subtropical and temperate regions. To clarify the genetic diversity of P. nicotianae and to understand its mode of transmission in Japan, we developed six new microsatellites markers, consisting of six loci and 39 alleles. In a phylogenetic analysis, 138 isolates, including 125 from Japan and 13 from overseas, were shown to differ, even though some were collected from the same host and location, suggesting that there is no geographic or host plant clustering. Population structure analysis also revealed a highly admixed population of P. nicotianae in Japan. Molecular analysis suggested high variance between individuals but no significant differences between populations. Both A1 and A2 mating types were present in the same population, which could be due to high levels of variance between individuals in the population. The absence of geographical structure between populations also suggests that the pathogen is able to migrate from one population to another. We propose that this phenomenon could result from human activities related to the transport of plant and associated agricultural materials.

Phytophthora cactorum, P. cinnamomi and P. lateralis were reported to be pathogenic on kiwifruit trees in the main planting areas of China. We attempted to simultaneously detect the three pathogens using a multiplex polymerase chain reaction (PCR) and to survey their occurrence in the main production areas. Because of the need to combine different primer pairs for the multiplex PCR and the low specificity of published specific primers for P. cactorum, P. cinnamomi and P. lateralis, new species-specific primers for the three species were designed based on the ras-related protein gene, Ypt1. The specificity of the designed primers was demonstrated using 52 isolates, including 44 Phytophthora species, three Pythium species, and three other soil-borne pathogens. A multiplex PCR method for the simultaneous detection of P. cactorum, P. cinnamomi and P. lateralis was established, and the three pathogens were detected in artificially and naturally infested soils, indicating that these markers can be used in the diagnosis of kiwifruit Phytophthora diseases. In a survey of these pathogens in the main kiwifruit planting areas of China, 99 soil samples were collected at different locations and in different seasons and subjected to the new method, and the distribution of the three pathogens in the main kiwifruit planting areas of China was determined.

The aim of the present study was to identify a strain of endophytic Bacillus species that control tomato bacterial wilt by foliar spray application. Fifty heat-tolerant endophytic bacteria were isolated from the surface-sterilized foliar tissues of symptomless tomato plants that had been pre-inoculated with the pathogen Ralstonia pseudosolanacearum. In the primary screening, we assessed the suppressive effects of a shoot-dipping treatment with bacterial strains against bacterial wilt on tomato seedlings grown on peat pellets. Bacillus sp. strains G1S3 and G4L1 significantly suppressed the incidence of tomato bacterial wilt. In subsequent pot experiments, the biocontrol efficacy of foliar spray application was examined under glasshouse conditions. G4L1 displayed consistent and significant disease suppression, and, thus, was selected as a biocontrol candidate. Moreover, the pathogen population in the stem of G4L1-treated plants was markedly smaller than that in control plants. A quantitative real-time PCR analysis revealed that the foliar spraying of tomato plants with G4L1 up-regulated the expression of PR-1a and LoxD in stem and GluB in roots upon the pathogen inoculation, implying that the induction of salicylic acid-, jasmonic acid-, and ethylene-dependent defenses was involved in the protective effects of this strain. In the re-isolation experiment, G4L1 efficiently colonized foliar tissues for at least 4‍ ‍weeks after spray application. Collectively, the present results indicate that G4L1 is a promising biocontrol agent for tomato bacterial wilt. Furthermore, to the best of our knowledge, this is the first study to report the biocontrol of bacterial wilt by the foliar spraying with an endophytic Bacillus species.

As global plant trade expands, tree disease epidemics caused by pathogen introductions are increasing. Since ca 2000, the introduced oomycete Phytophthora ramorum has caused devastating epidemics in Europe and North America, spreading as four ancient clonal lineages, each of a single mating type, suggesting different geographical origins. We surveyed laurosilva forests for P. ramorum around Fansipan mountain on the Vietnam-China border and on Shikoku and Kyushu islands, southwest Japan. The surveys yielded 71 P. ramorum isolates which we assigned to eight new lineages, IC1 to IC5 from Vietnam and NP1 to NP3 from Japan, based on differences in colony characteristics, gene x environment responses and multigene phylogeny. Molecular phylogenetic trees and networks revealed the eight Asian lineages were dispersed across the topology of the introduced European and North American lineages. The deepest node within P. ramorum, the divergence of lineages NP1 and NP2, was estimated at 0.5 to 1.6 Myr. The Asian lineages were each of a single mating type, and at some locations, lineages of “opposite” mating type were present, suggesting opportunities for inter-lineage recombination. Based on the high level of phenotypic and phylogenetic diversity in the sample populations, the coalescence results and the absence of overt host symptoms, we conclude that P. ramorum comprises many anciently divergent lineages native to the laurosilva forests between eastern Indochina and Japan.

Hydrogen peroxide (H2O2) is an important signaling molecule for various physiological processes that take place in higher plants during environmental adaptation. This article addresses the role of H2O2 in signal transduction and gene regulation in the context of a variety of biotic and abiotic stresses. Based on a comparative analysis using public and our own transcriptome data, we consider H2O2 as an independent mediator that acts in response to any of the plant stresses and also as having a role in the synergetic response to combinations of stress factors. Here, we propose H2O2 as a signal molecule that is able to trigger gene activation of a conserved set of genes that make a universal transcriptional subnetwork comprising tens of transcription factors.

The plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2 (GP17-2), induces systemic resistance against Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis thaliana. The molecular mechanisms underlying induced systemic resistance (ISR) by GP17-2 were investigated in the present study. Microscopic observations revealed that stomatal reopening by Pst was restricted by elicitation with the culture filtrate (CF) from GP17-2. A gene expression analysis of MYB44, which enhances abscisic acid signaling and consequently closes stomata, revealed that the gene was activated by CF. CF-elicited myb44 mutant plants failed to restrict stomatal reopening and showed lower resistance to Pst than wild-type plants. These results indicate that stomatal resistance by GP17-2 is mediated by the gene activation of MYB44. We herein revealed that the MYB44-mediated prevention of penetration through the stomata is one of the components responsible for GP17-2-elicited ISR.

Pyrenochaeta lycopersici is the causal agent of corky root, an important soilborne disease of tomato (Lycopersicon esculentum) and other solanaceous crops. P. lycopersici isolates are classified into Types 1 and 2 on the basis of several physiological and molecular features. In this study, we aimed to establish a loop-mediated isothermal amplification (LAMP) method to identify Types 1 and 2 isolates. Using specific LAMP primer sets for Types 1 and 2, both types were easily identified within 35 min. The LAMP method demonstrated equal sensitivity to the polymerase chain reaction in molecular identification of the pathogen in cultured mycelia, infested plant roots, and their surrounding soil. The LAMP technology will contribute to the detection and identification of the pathogen, crop protection, and thorough understanding about the ecology of corky root disease of tomato.

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of ‘4 bases—a spacer with a flexible length—4 bases’. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein–DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic- and abiotic-induced redox signals. RRTF1 is highly conserved in angiosperms, but its physiological role remains elusive. Here we show that inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Transgenic lines overexpressing RRTF1 are impaired in root and shoot development, light sensitive, and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica, which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors that scavenge ROS. More than 800 genes were detected in mature leaves and seedlings of transgenic lines overexpressing RRTF1; ∼ 40% of them have stress-, redox-, ROS-regulated-, ROS-scavenging-, defense-, cell death- and senescence-related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box-like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli and H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains a GCC-box-like sequence in its promoter, but transgenic lines overexpressing RAP2.6 do not accumulate higher ROS levels. RRTF1 also stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the elevated levels of the highly conserved RRTF1 induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals.

Hydrogen peroxide acts as a signaling molecule mediating the acquisition of tolerance to both biotic and abiotic stresses. Identification of marker genes for H2O2 response could help to intercept the signaling network of stress response of plants. Here, we describe application of marker genes for H2O2 responses to monitoring several abiotic stress responses. Arabidopsis plants were treated with UV-B, high light, and cold stresses, where involvement of H2O2-mediated signaling is known or suggested. Monitoring of these stress responses with molecular markers using quantitative real-time RT-PCR can detect landmark events in the sequential stress responses. These methods can be used for analysis of mutants and transgenic plants to examine natural H2O2 responses that are involved in environmental adaptation.

Hydrogen peroxide acts as a signaling molecule mediating the acquisition of tolerance to both biotic and abiotic stresses. Identification of marker genes for H2O2 response could help to intercept the signaling network of stress response of plants. Here, we describe application of marker genes for H2O2 responses to monitoring several abiotic stress responses. Arabidopsis plants were treated with UV-B, high light, and cold stresses, where involvement of H2O2-mediated signaling is known or suggested. Monitoring of these stress responses with molecular markers using quantitative real-time RT-PCR can detect landmark events in the sequential stress responses. These methods can be used for analysis of mutants and transgenic plants to examine natural H2O2 responses that are involved in environmental adaptation.