Survival dynamics of stick insect and the impact of environmental factors on natural fungal infection during the rainy season.

Phasmatodea, commonly known as stick insects, are recognized as noteworthy pests globally, impacting agriculture and forest ecosystems. Among them, the outbreak of Ramulus mikado has emerged as a notable concern in East Asian forests. Recently, Metarhizium phasmatodeae has been identified as utilizing stick insects as hosts. We have observed evidence of this entomopathogenic fungus infecting stick insects. Given the increase in these occurrences during the rainy period, this study investigated the relationship between the survival of R. mikado and the M. phasmatodeae infection during the rainy seasons of 2022 and 2023. We collected stick insects in two representative forests of the Republic of Korea and examined insect survival, fungal infection, and various environmental factors. No infections were detected in specimens collected in June before the rainy season, but from July onwards, both the mortality of R. mikado and the fungal infection substantially increased. By the last sampling date of each year, 75% (2022), 71.4% (2023) of the specimens were infected, and over 90% of the total individuals succumbed as a result. Fungi isolated from deceased R. mikado were successfully identified as M. phasmatodeae using morphological and taxonomic approaches. Various statistical analyses, including principal component analysis and modeling, revealed a robust association between fungal infection and the survival of stick insects. The results highlight the correlation between mass deaths of stick insects and fungal infection, particularly during the summer rainy season. These findings offer valuable insights for forecasting R. mikado population in the upcoming year and developing effective pest control strategies.

Past and Future Epidemiological Perspectives and Integrated Management of Rice Bakanae in Korea.

In the past, rice bakanae was considered an endemic disease that did not cause significant losses in Korea; however, the disease has recently become a serious threat due to climate change, changes in farming practices, and the emergence of fungicide-resistant strains. Since the bakanae outbreak in 2006, its incidence has gradually decreased due to the application of effective control measures such as hot water immersion methods and seed disinfectants. However, in 2013, a marked increase in bakanae incidence was observed, causing problems for rice farmers. Therefore, in this review, we present the potential risks from climate change based on an epidemiological understanding of the pathogen, host plant, and environment, which are the key elements influencing the incidence of bakanae. In addition, disease management options to reduce the disease pressure of bakanae below the economic threshold level are investigated, with a specific focus on resistant varieties, as well as chemical, biological, cultural, and physical control methods. Lastly, as more effective countermeasures to bakanae, we propose an integrated disease management option that combines different control methods, including advanced imaging technologies such as remote sensing. In this review, we revisit and examine bakanae, a traditional seed-borne fungal disease that has not gained considerable attention in the agricultural history of Korea. Based on the understanding of the present significance and anticipated risks of the disease, the findings of this study are expected to provide useful information for the establishment of an effective response strategy to bakanae in the era of climate change.

Adapting to the projected epidemics of Fusarium head blight of wheat in Korea under climate change scenarios.

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum Schwabe, is an emerging threat to wheat production in Korea under a changing climate. The disease occurrence and accumulation of associated trichothecene mycotoxins in wheat kernels strongly coincide with warm and wet environments during flowering. Recently, the International Panel for Climate Change released the 6th Coupled Model Intercomparison Project (CMIP6) climate change scenarios with shared socioeconomic pathways (SSPs). In this study, we adopted GIBSIM, an existing mechanistic model developed in Brazil to estimate the risk infection index of wheat FHB, to simulate the potential FHB epidemics in Korea using the SSP245 and SSP585 scenarios of CMIP6. The GIBSIM model simulates FHB infection risk from airborne inoculum density and infection frequency using temperature, precipitation, and relative humidity during the flowering period. First, wheat heading dates, during which GIBSIM runs, were predicted over suitable areas of winter wheat cultivation using a crop development rate model for wheat phenology and downscaled SSP scenarios. Second, an integrated model combining all results of wheat suitability, heading dates, and FHB infection risks from the SSP scenarios showed a gradual increase in FHB epidemics towards 2100, with different temporal and spatial patterns of varying magnitudes depending on the scenarios. These results indicate that proactive management strategies need to be seriously considered in the near future to minimize the potential impacts of the FHB epidemic under climate change in Korea. Therefore, available wheat cultivars with early or late heading dates were used in the model simulations as a realistic adaptation measure. As a result, wheat cultivars with early heading dates showed significant decreases in FHB epidemics in future periods, emphasizing the importance of effective adaptation measures against the projected increase in FHB epidemics in Korea under climate change.

Optimizing Artificial Neural Network-Based Models to Predict Rice Blast Epidemics in Korea.

To predict rice blast, many machine learning methods have been proposed. As the quality and quantity of input data are essential for machine learning techniques, this study develops three artificial neural network (ANN)-based rice blast prediction models by combining two ANN models, the feed-forward neural network (FFNN) and long short-term memory (LSTM), with diverse input datasets, and compares their performance. The Blast_Weather_FFNN model had the highest recall score (66.3%) for rice blast prediction. This model requires two types of input data: blast occurrence data for the last 3 years and weather data (daily maximum temperature, relative humidity, and precipitation) between January and July of the prediction year. This study showed that the performance of an ANN-based disease prediction model was improved by applying suitable machine learning techniques together with the optimization of hyperparameter tuning involving input data. Moreover, we highlight the importance of the systematic collection of long-term disease data.

Comparison of projected rice blast epidemics in the Korean Peninsula between the CMIP5 and CMIP6 scenarios.

Recently, the International Panel for Climate Change released the 6th Coupled Model Intercomparison Project (CMIP6) climate change scenarios with shared socioeconomic pathways (SSPs). The SSP scenarios result in significant changes to climate variables in climate projections compared to their predecessor, the representative concentration pathways from the CMIP5. Therefore, it is necessary to examine whether the CMIP6 scenarios differentially impact plant-disease ecosystems compared to the CMIP5 scenarios. In this study, we used the EPIRICE-LB model to simulate and compare projected rice blast disease epidemics in the Korean Peninsula using five selected family global climate models (GCMs) of the CMIP5 and CMIP6 for two forcing scenarios. We found a similar decrease in rice blast epidemics in both CMIP scenarios; however, this decrease was greater in the CMIP6 scenarios. In addition, distinctive epidemic trends were found in North Korea, where the rice blast epidemics increase until the mid-2040s but decrease thereafter until 2100, with different spatial patterns of varying magnitudes. Controlling devastating rice blast diseases will remain important during the next decades in North Korea, where appropriate chemical controls are unavailable due to chronic economic and political issues. Overall, our analyses using the new CMIP6 scenarios reemphasized the importance of developing effective control measures against rice blast for specific high-risk areas and the need for a universal impact and vulnerability assessment platform for plant-disease ecosystems that can be used with new climate change scenarios in the future. Supplementary information: The online version contains supplementary material available at 10.1007/s10584-022-03410-2.

Retrospective Study on the Seasonal Forecast-Based Disease Intervention of the Wheat Blast Outbreaks in Bangladesh.

Seasonal disease risk prediction using disease epidemiological models and seasonal forecasts has been actively sought over the last decades, as it has been believed to be a key component in the disease early warning system for the pre-season planning of local or national level disease control. We conducted a retrospective study using the wheat blast outbreaks in Bangladesh, which occurred for the first time in Asia in 2016, to study a what-if scenario that if there was seasonal disease risk prediction at that time, the epidemics could be prevented or reduced through prediction-based interventions. Two factors govern the answer: the seasonal disease risk prediction is accurate enough to use, and there are effective and realistic control measures to be used upon the prediction. In this study, we focused on the former. To simulate the wheat blast risk and wheat yield in the target region, a high-resolution climate reanalysis product and spatiotemporally downscaled seasonal climate forecasts from eight global climate models were used as inputs for both models. The calibrated wheat blast model successfully simulated the spatial pattern of disease epidemics during the 2014-2018 seasons and was subsequently used to generate seasonal wheat blast risk prediction before each winter season starts. The predictability of the resulting predictions was evaluated against observation-based model simulations. The potential value of utilizing the seasonal wheat blast risk prediction was examined by comparing actual yields resulting from the risk-averse (proactive) and risk-disregarding (conservative) decisions. Overall, our results from this retrospective study showed the feasibility of seasonal forecast-based early warning system for the pre-season strategic interventions of forecasted wheat blast in Bangladesh.

Development of a Daily Epidemiological Model of Rice Blast Tailored for Seasonal Disease Early Warning in South Korea.

Early warning services for crop diseases are valuable when they provide timely forecasts that farmers can utilize to inform their disease management decisions. In South Korea, collaborative disease controls that utilize unmanned aerial vehicles are commonly performed for most rice paddies. However, such controls could benefit from seasonal disease early warnings with a lead time of a few months. As a first step to establish a seasonal disease early warning service using seasonal climate forecasts, we developed the EPIRICE Daily Risk Model for rice blast by extracting and modifying the core infection algorithms of the EPIRICE model. The daily risk scores generated by the EPIRICE Daily Risk Model were successfully converted into a realistic and measurable disease value through statistical analyses with 13 rice blast incidence datasets, and subsequently validated using the data from another rice blast experiment conducted in Icheon, South Korea, from 1974 to 2000. The sensitivity of the model to air temperature, relative humidity, and precipitation input variables was examined, and the relative humidity resulted in the most sensitive response from the model. Overall, our results indicate that the EPIRICE Daily Risk Model can be used to produce potential disease risk predictions for the seasonal disease early warning service.

The First Finding of the Lichen Solorina saccata at an Algific Talus Slope in Korea.

An algific talus slope is composed of broken rocks with vents connected to an ice cave, releasing cool air in summer and relatively warmer air in winter to maintain a more stable microclimate all year round. Such geological features create a very unusual and delicate ecosystem. Although there are around 25 major algific talus slopes in Korea, lichen ecology of these areas had not been investigated to date. In this study, we report the first exploration of lichen diversity and ecology at an algific talus slope, Jangyeol-ri, in Korea. A total of 37 specimens were collected over 2017-2018. Morphological and sequencing analysis revealed 27 species belonging to 18 genera present in the area. Of particular interest among these species was Solorina saccata, as it has previously not been reported in Korea and most members of genus Solorina are known to inhabit alpine regions of the Northern Hemisphere. We provide here a taxonomic key for S. saccata alongside molecular phylogenetic analyses and prediction of potential habitats in South Korea. Furthermore, regions in South Korea potentially suitable for Solorina spp. were predicted based on climatic features of known habitats around the globe. Our results showed that the suitable areas are mostly at high altitudes in mountainous areas where the annual temperature range does not exceed 26.6 °C. Further survey of other environmental conditions determining the suitability of Solorina spp. should lead to a more precise prediction of suitable habitats and trace the origin of Solorina spp. in Korea.

An Integrated Modeling Approach for Predicting Potential Epidemics of Bacterial Blossom Blight in Kiwifruit under Climate Change.

The increasing variation in climatic conditions under climate change directly influences plant-microbe interactions. To account for as many variables as possible that may play critical roles in such interactions, the use of an integrated modeling approach is necessary. Here, we report for the first time a local impact assessment and adaptation study of future epidemics of kiwifruit bacterial blossom blight (KBB) in Jeonnam province, Korea, using an integrated modeling approach. This study included a series of models that integrated both the phenological responses of kiwifruit and the epidemiological responses of KBB to climatic factors with a 1 km resolution, under the RCP8.5 climate change scenario. Our results indicate that the area suitable for kiwifruit cultivation in Jeonnam province will increase and that the flowering date of kiwifruit will occur increasingly earlier, mainly due to the warming climate. Future epidemics of KBB during the predicted flowering periods were estimated using the Pss-KBB Risk Model over the predicted suitable cultivation regions, and we found location-specific, periodic outbreaks of KBB in the province through 2100. Here, we further suggest a potential, scientifically-informed, long-term adaptation strategy using a cultivar of kiwifruit with a different maturity period to relieve the pressures of future KBB risk. Our results clearly show one of the possible options for a local impact assessment and adaptation study using multiple models in an integrated way.

Wheat Blast in Bangladesh: The Current Situation and Future Impacts.

Wheat blast occurred in Bangladesh for the first time in Asia in 2016. It is caused by a fungal pathogen, Magnaporthe oryzae Triticum (MoT) pathotype. In this review, we focused on the current status of the wheat blast in regard to host, pathogen, and environment. Despite the many efforts to control the disease, it expanded to neighboring regions including India, the world's second largest wheat producer. However, the disease occurrence has definitely decreased in quantity, because of many farmers chose to grow alternate crops according to the government's directions. Bangladesh government planned to introduce blast resistant cultivars but knowledges about genetics of resistance is limited. The genome analyses of the pathogen population revealed that the isolates caused wheat blast in Bangladesh are genetically close to a South American lineage of Magnaporthe oryzae. Understanding the genomes of virulent strains would be important to find target resistance genes for wheat breeding. Although the drier winter weather in Bangladesh was not favorable for development of wheat blast before, recent global warming and climate change are posing an increasing risk of disease development. Bangladesh outbreak in 2016 was likely to be facilitated by an extraordinary warm and humid weather in the affected districts before the harvest season. Coordinated international collaboration and steady financial supports are needed to mitigate the fearsome wheat blast in South Asia before it becomes a catastrophe.

Outbreak and Spread of Bacterial Canker of Kiwifruit Caused by Pseudomonas syringae pv. actinidiae Biovar 3 in Korea.

A bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), is a causal agent of kiwifruit bacterial canker worldwide. Psa biovar 3 (Psa3) was first detected in 2011 at an orchard in Dodeok-myeon, Goheunggun, Jeonnam Province in Korea. In this study, we present the results of an epidemiological study regarding Psa3 occurrence on kiwifruit orchards in Korea for the period of 2013 to 2015. Since the first detection of Psa3 in 2011, there was no further case reported by 2013. However, Psa3 was rapidly spreading to 33 orchards in 2014; except for three orchards in Sacheonsi, Gyeongnam Province, most cases were reported in Jeju Island. Entering 2015, bacterial canker by Psa3 became a pandemic in Korea, spreading to 72 orchards in Jeju Island, Jeonnam, and Gyeongnam Provinces. Our epidemiological study indicated that the first Psa3 incidence in 2011 might result from an introduction of Psa3 through imported seedlings from China in 2006. Apart from this, it was estimated that most Psa3 outbreaks from 2014 to 2015 were caused by pollens imported from New Zealand and China for artificial pollination. Most kiwifruit cultivars growing in Korea were infected with Psa3; yellow-fleshed cultivars (Yellow-king, Hort16A, Enza-gold, Zecy-gold, and Haegeum), red-fleshed cultivars (Hongyang and Enza-Red), green-fleshed cultivars (Hayward and Daeheung), and even a kiwiberry (Skinny-green). However, susceptibility to canker differed among cultivars; yellow- and red-fleshed cultivars showed much more severe symptoms compared to the green-fleshed cultivars of kiwifruit and a kiwiberry.

Outbreaks of Yuzu Dieback in Goheung Area: Possible Causes Deduced from Weather Extremes.

Starting in 2012, severe diebacks usually accompanied by abundant gum exudation have occurred on yuzu trees in Goheung-gun, Jeonnam Province, where severely affected trees were occasionally killed. On-farm surveys were conducted at 30 randomly-selected orchards located at Pungyang-myeon, Goheung-gun, and the resulting disease incidences were 18.5% and 39.6% for dieback and gumming symptoms, respectively. Black spots on branches and leaves also appeared on infected trees showing a typical dieback symptom. Morphological and molecular identifications of the isolated fungal organisms from lesions on the symptomatic leaves and branches revealed that they are identical to Phomopsis citri, known to cause gummosis. In order to find the reason for this sudden epidemic, we investigated the weather conditions that are exclusively distinct from previous years, hypothesizing that certain weather extremes might have caused the severe induction of pre-existing disease for yuzu. There were two extreme temperature drops beyond the yuzu's cold hardiness limit right after an abnormally-warm-temperature-rise during the winter of 2011-12, which could cause severe frost damage resulting in mechanical injuries and physiological weakness to the affected trees. Furthermore, there was an increased frequency of strong wind events, seven times in 2012 compared to only a few times in the previous years, that could also lead to extensive injuries on branches. In conclusion, we estimated that the possible damages by severe frost and frequent strong wind events during 2012 could cause the yuzu trees to be vulnerable to subsequent fungal infection by providing physical entries and increasing plant susceptibility to infections.

TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen.

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus DeltatmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola DeltatmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DeltatmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola DeltatmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DeltatmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola DeltatmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

Biosynthesis and role in virulence of the histone deacetylase inhibitor depudecin from Alternaria brassicicola.

Depudecin, an eleven-carbon linear polyketide made by the pathogenic fungus Alternaria brassicicola, is an inhibitor of histone deacetylase (HDAC). A chemically unrelated HDAC inhibitor, HC toxin, was earlier shown to be a major virulence factor in the interaction between Cochliobolus carbonum and its host, maize. In order to test whether depudecin is also a virulence factor for A. brassicicola, we identified the genes for depudecin biosynthesis and created depudecin-minus mutants. The depudecin gene cluster contains six genes (DEP1 to DEP6), which are predicted to encode a polyketide synthase (AbPKS9 or DEP5), a transcription factor (DEP6), two monooxygenases (DEP2 and DEP4), a transporter of the major facilitator superfamily (DEP3), and one protein of unknown function (DEP1). The involvement in depudecin production of DEP2, DEP4, DEP5, and DEP6 was demonstrated by targeted gene disruption. DEP6 is required for expression of DEP1 through DEP5 but not the immediate flanking genes, thus defining a coregulated depudecin biosynthetic cluster. The genes flanking the depudecin gene cluster but not the cluster itself are conserved in the same order in the related fungi Stagonospora nodorum and Pyrenophora tritici-repentis. Depudecin-minus mutants have a small (10%) but statistically significant reduction in virulence on cabbage (Brassica oleracea) but not on Arabidopsis. The role of depudecin in virulence is, therefore, less dramatic than that of HC toxin.

Identification of novel virulence factors associated with signal transduction pathways in Alternaria brassicicola.

Alternaria brassicicola is an important, necrotrophic fungal pathogen that causes black spot disease on Brassicas. In order to study pathogenicity mechanisms, gene deletion mutants were generated for 21 putative regulatory genes including kinases and transcription factors subjectively selected from the annotated A. brassicicola genome. Except for Ste12, the deletion of the SNF1 kinase, XlnR, and CreA homologues that control cell wall-degrading enzyme production did not significantly affect virulence in contrast to other pathogenic fungi. Only deletion of XlnR but not CreA, Ste12 or SNF1 impaired the fungus' ability to utilize sole carbon sources suggesting Alternaria regulates expression of cell wall-degrading enzymes in a novel manner. In addition, two novel virulence factors encoding a transcription factor (AbPro1) and a two-component histidine kinase gene (AbNIK1) were discovered. Deletion of AbPro1 resulted in a 70% reduction in virulence and a 25% reduction in vegetative growth rates in vitro. Deletion of AbNIK1 resulted in a near complete loss of virulence, increased sensitivity to osmotic stress, and no changes in vegetative growth rates in vitro. Interestingly, addition of long polypeptides to spores of both Deltaabste12 and Deltaabnik1 during inoculations resulted in a complete restoration of pathogenicity through a yet to be defined mechanism.

A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus.

At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola.

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.

Functional analysis of the Alternaria brassicicola non-ribosomal peptide synthetase gene AbNPS2 reveals a role in conidial cell wall construction.

SUMMARY Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2. The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae, AbrePsy1, Cochliobolus heterostrophus, NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo, and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence.

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.

Structural Modifications and Programmed Cell Death of Chili Pepper Fruit Related to Resistance Responses to Colletotrichum gloeosporioides Infection.

ABSTRACT Postharvest (detached) and in planta (attached) fruits of pepper plants, Capsicum annuum cv. Jejujaerae (susceptible) and Capsicum baccatum cv. PBC80 (resistant), inoculated with the anthracnose pathogen Colletotrichum gloeosporioides were examined using light, confocal laser scanning, and electron microscopy to compare the cytological differences between the compatible and incompatible interactions. In nonwound inoculation of postharvest pepper fruit, resistant pepper tissues showed a significant increase in the thickness of the cuticle layer compared with that of the susceptible and noninoculated fruit. Cytological features of programmed cell death (PCD) were observed in the resistant pepper fruit with postharvest inoculation, and these were characterized by positive responses to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The oligonucleosomal fragments of DNA were confirmed electrophoretically as DNA laddering. The PCD-positive responses occurred around the inoculation sites early in in planta wound inoculation in the resistant pepper. Nuclear modifications and structural changes of hypersensitivity were also observed in the resistant fruit, including separation of the plasma membrane from the cell wall, dilation of the endoplasmic reticulum, accumulation of electron-dense inclusions in vacuoles, and cytoplasmic vacuolization accompanying fragmentation of the cytoplasm. These structural changes may also implicate PCD-like host responses. In addition, in planta wound inoculation resulted in cell enlargement and cell division during the later stages of infection to form a periderm-like boundary layer around the inoculation site.