Stalk rot species diversity and molecular phylogeny associated with diseased maize in India.

Stalk rot disease is a major constraint in maize production and till date reported to be caused by two to three species of phytopathogenic fungi but, in our present study, we disclose the first report of stalk rot is caused by complex species of phytopathogens, which belongs to five different genera. Therefore, to substantiate these findings, a total of 105 diseased samples of maize were collected from 21 different locations in six different geographical locations of India from which 48 isolates were used for the research study. Morphological features such as pigmentation, colony color, type of mycelium and pattern of mycelium was examined using macro and microscopic methods. A total of 11 different spp. of pathogens belonging to the five different genera: Fusarium verticillioides (56.25%), F. equiseti (14.5%), F. andiyazi (6.25%), F. solani (2.08%), F. proliferatum (2.08%), F. incarnatum (2.08%), Lasidioplodia theobrame (6.25%), Exserohilum rostrtum (4.16%), Nigrospora spp. (4.16%). and Schizophyllum commune (2.08%) were identified by different housekeeping genes (ITS, TEF-1α, RPB2 and Actin). Fusarium verticillioides, F. equiseti and F. andiyazi were major pathogens involved in stalk rot. This is the first report on F. proliferatum, F. solani, F. incarnatum, Lasidioplodia theobrame, Exserohilum rostrtum, Nigrospora spp. and Schizophyllum commune causing stalk rot of maize and their distribution in the different states of India. Studies on population dynamics of PFSR will enhance the understanding of pathogen behavior, virulence, or its association with different pathogens across India, which will facilitate the development of resistant maize genotypes against the PFSR.

Detection of Latent Peronospora effusa Infections in Spinach.

Downy mildew disease of spinach, caused by Peronospora effusa, is managed in conventional fields by a combination of host resistance and scheduled fungicide applications. Fungicides are currently applied to prevent downy mildew epidemics regardless of the infection status of spinach crops. A more streamlined approach would be to develop methods to target either latent infections for fungicide application in conventional production systems or to hasten harvest in organic production. In this study, conventional polymerase chain reaction (PCR) was applied to detect P. effusa DNA in symptomless spinach leaves in three spatially and temporally separated field plots, each containing four 2-m beds, 35 m in length. Spinach leaves were sampled weekly at 3-m intervals at 48 locations throughout each plot. Initial samples were asymptomatic and yet PCR enabled detection of P. effusa DNA extracted from sampled spinach leaves. Detection of latent downy mildew infection in spinach leaves was confirmed by PCR as early as 7 days prior to symptom development. The limit of pathogen DNA detection in spinach leaves was calculated at 10 pg using the conventional PCR approach. Quantitative PCR with TaqMan methodology revealed the presence of inhibitors from spinach leaf DNA extracts and affected amplification efficiencies, but not when diluted, enabling detection of P. effusa DNA at a concentration of <0.1 pg. In conclusion, detection of latent infections may enable management decisions for earlier-than-normal harvest of infected, symptomless organic crops, and for timing fungicide applications on symptomless plants in conventional production.

Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR.

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.

Identification of Phakopsora pachyrhizi Candidate Effectors with Virulence Activity in a Distantly Related Pathosystem.

Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, a disease that causes enormous economic losses, most markedly in South America. P. pachyrhizi is a biotrophic pathogen that utilizes specialized feeding structures called haustoria to colonize its hosts. In rusts and other filamentous plant pathogens, haustoria have been shown to secrete effector proteins into their hosts to permit successful completion of their life cycle. We have constructed a cDNA library from P. pachyrhizi haustoria using paramagnetic bead-based methodology and have identified 35 P. pachyrhizi candidate effector (CE) genes from this library which are described here. In addition, we quantified the transcript expression pattern of six of these genes and show that two of these CEs are able to greatly increase the susceptibility of Nicotiana benthamiana to Phytophthora infestans. This strongly suggests that these genes play an important role in P. pachyrhizi virulence on its hosts.

Plasmolysis and Vital Staining Reveal Viable Oospores of Peronospora effusa in Spinach Seed Lots.

Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seedborne transmission of this pathogen has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment was examined by microscopy. Of the analyzed seed lots, 16% were positive for oospores and an additional 6% for sporangiophores characteristic of P. effusa. Application of a P. effusa-specific quantitative polymerase chain reaction assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots that were proven to carry the pathogen from the above tests was tested using two independent methods, one involving plasmolysis and the other trypan blue staining. The oospores plasmolyzed in 4 M sodium chloride and were deplasmolyzed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and suggest that the transmission of viable oospores of P. effusa in spinach seed does occur. Elimination of the pathogen on seed, in addition to other management approaches, will be useful in reducing the extent and severity of downy mildew on spinach crops and diminishing pathogen spread through seed.

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum.

BACKGROUND: Small non-coding RNAs (sRNA) are emerging as major components of the cell's regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs. RESULTS: Using RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box &solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq. CONCLUSIONS: Our results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.

Transcriptomics of the rice blast fungus Magnaporthe oryzae in response to the bacterial antagonist Lysobacter enzymogenes reveals candidate fungal defense response genes.

Plants and animals have evolved a first line of defense response to pathogens called innate or basal immunity. While basal defenses in these organisms are well studied, there is almost a complete lack of understanding of such systems in fungal species, and more specifically, how they are able to detect and mount a defense response upon pathogen attack. Hence, the goal of the present study was to understand how fungi respond to biotic stress by assessing the transcriptional profile of the rice blast pathogen, Magnaporthe oryzae, when challenged with the bacterial antagonist Lysobacter enzymogenes. Based on microscopic observations of interactions between M. oryzae and wild-type L. enzymogenes strain C3, we selected early and intermediate stages represented by time-points of 3 and 9 hours post-inoculation, respectively, to evaluate the fungal transcriptome using RNA-seq. For comparative purposes, we also challenged the fungus with L. enzymogenes mutant strain DCA, previously demonstrated to be devoid of antifungal activity. A comparison of transcriptional data from fungal interactions with the wild-type bacterial strain C3 and the mutant strain DCA revealed 463 fungal genes that were down-regulated during attack by C3; of these genes, 100 were also found to be up-regulated during the interaction with DCA. Functional categorization of genes in this suite included those with roles in carbohydrate metabolism, cellular transport and stress response. One gene in this suite belongs to the CFEM-domain class of fungal proteins. Another CFEM class protein called PTH11 has been previously characterized, and we found that a deletion in this gene caused advanced lesion development by C3 compared to its growth on the wild-type fungus. We discuss the characterization of this suite of 100 genes with respect to their role in the fungal defense response.

RNA-Seq reveals infection-related global gene changes in Phytophthora phaseoli, the causal agent of lima bean downy mildew.

Lima bean is an important vegetable processing crop to the mid-Atlantic USA and is highly susceptible to the oomycete pathogen Phytophthora phaseoli, which causes downy mildew. Genetic resistance and fungicides are used to manage P. phaseoli and often fail. Currently, the molecular basis of the interaction between this host and pathogen is unknown. To begin to rectify this situation, we used Illumina RNA-Seq to perform a global transcriptome analysis comparing P. phaseoli growing in culture with P. phaseoli infecting its host. Sequence reads from a total of six libraries mapped to gene models from the closely related late blight pathogen, Phytophthora infestans, resulting in 10 427 P. phaseoli genes with homology to P. infestans and expression in at least one library. Of these, 318 P. phaseoli homologues matched known or putative virulence genes in P. infestans. Two well-studied classes, RxLRs and elicitins, were up-regulated in planta, whereas the reverse was true for another class, called crinklers. These results are discussed with respect to the differences and similarities in the pathogenicity mechanisms of P. phaseoli and P. infestans.

Genome evolution following host jumps in the Irish potato famine pathogen lineage.

Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.

The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana.

The majority of plant growth promoting rhizobacteria (PGPR) confer plant immunity against a wide range of foliar diseases by activating plant defences that reduce a plant's susceptibility to pathogen attack. Here we show that Arabidopsis thaliana (Col-0) plants exposed to Bacillus subtilis strain FB17 (hereafter FB17), results in reduced disease severity against Pseudomonas syringae pv. tomato DC3000 (hereafter DC3000) compared to plants without FB17 treatment. Exogenous application of the B. subtilis derived elicitor, acetoin (3-hydroxy-2-butanone), was found to trigger induced systemic resistance (ISR) and protect plants against DC3000 pathogenesis. Moreover, B. subtilis acetoin biosynthetic mutants that emitted reduced levels of acetoin conferred reduced protection to A. thaliana against pathogen infection. Further analysis using FB17 and defense-compromised mutants of A. thaliana indicated that resistance to DC3000 occurs via NPR1 and requires salicylic acid (SA)/ethylene (ET) whereas jasmonic acid (JA) is not essential. This study provides new insight into the role of rhizo-bacterial volatile components as elicitors of defense responses in plants.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.