Epidemiological Criteria to Support Breeding Tactics Against the Emerging, High-Consequence Wheat Blast Disease.

Plant disease epidemiology can make a significant contribution for cultivar selection by elucidating the principles of an epidemic under different levels of resistance. For emerging diseases as wheat blast (WB), epidemiological parameters can provide support for better selection of genetic resources. Field experiments were conducted at two locations in Bolivia in 2018-2019 to characterize the temporal dynamics of the disease on 10 cultivars with different levels of reaction to WB. Logistic models best (R2 = 0.70-0.96) fit the disease progress curve in all cultivars followed by Gompertz (R2 = 0.64-0.94), providing additional evidence of a polycyclic disease. Total area under disease progress curve (tAUDPC), final disease severity (Ymax), and logistic apparent infection rates (rL*) were shown to be appropriate epidemiological parameters for describing resistance and cultivar selection. Cultivars that showed a high spike AUDPC (sAUDPC) showed a high leaf AUDPC (lAUDPC). tAUPDC, Ymax, and rL* were positively correlated among them (P < 0.01) and all were negatively correlated with grain weight (P < 0.01). Based on the epidemiological parameters used, cultivars that showed resistance to WB were Urubó, San Pablo, and AN-120, which were previously reported to have effective resistance against the disease under field conditions. The information generated could help breeding programs to make technical decisions about relevant epidemiological parameters to consider prior to cultivar release.

Temporal Dynamics of Wheat Blast Epidemics and Disease Measurements Using Multispectral Imagery.

Wheat blast is a devastating disease caused by the Triticum pathotype of Magnaporthe oryzae. M. oryzae Triticum is capable of infecting leaves and spikes of wheat. Although symptoms of wheat spike blast (WSB) are quite distinct in the field, symptoms on leaves (WLB) are rarely reported because they are usually inconspicuos. Two field experiments were conducted in Bolivia to characterize the change in WLB and WSB intensity over time and determine whether multispectral imagery can be used to accurately assess WSB. Disease progress curves (DPCs) were plotted from WLB and WSB data, and regression models were fitted to describe the nature of WSB epidemics. WLB incidence and severity changed over time; however, the mean WLB severity was inconspicuous before wheat began spike emergence. Overall, both Gompertz and logistic models helped to describe WSB intensity DPCs fitting classic sigmoidal shape curves. Lin's concordance correlation coefficients were estimated to measure agreement between visual estimates and digital measurements of WSB intensity and to estimate accuracy and precision. Our findings suggest that the change of wheat blast intensity in a susceptible host population over time does not follow a pattern of a monocyclic epidemic. We have also demonstrated that WSB severity can be quantified using a digital approach based on nongreen pixels. Quantification was precise (0.96 < r> 0.83) and accurate (0.92 < ρ > 0.69) at moderately low to high visual WSB severity levels. Additional sensor-based methods must be explored to determine their potential for detection of WLB and WSB at earlier stages.

The 2NS Translocation from Aegilops ventricosa Confers Resistance to the Triticum Pathotype of Magnaporthe oryzae.

Wheat blast is a serious disease caused by the fungus Magnaporthe oryzae (Triticum pathotype) (MoT). The objective of this study was to determine the effect of the 2NS translocation from Aegilops ventricosa (Zhuk.) Chennav on wheat head and leaf blast resistance. Disease phenotyping experiments were conducted in growth chamber, greenhouse, and field environments. Among 418 cultivars of wheat (Triticum aestivum L.), those with 2NS had 50.4 to 72.3% less head blast than those without 2NS when inoculated with an older MoT isolate under growth chamber conditions. When inoculated with recently collected isolates, cultivars with 2NS had 64.0 to 80.5% less head blast. Under greenhouse conditions when lines were inoculated with an older MoT isolate, those with 2NS had a significant head blast reduction. With newer isolates, not all lines with 2NS showed a significant reduction in head blast, suggesting that the genetic background and/or environment may influence the expression of any resistance conferred by 2NS. However, when near-isogenic lines (NILs) with and without 2NS were planted in the field, there was strong evidence that 2NS conferred resistance to head blast. Results from foliar inoculations suggest that the resistance to head infection that is imparted by the 2NS translocation does not confer resistance to foliar disease. In conclusion, the 2NS translocation was associated with significant reductions in head blast in both spring and winter wheat.

[Ambulatory blood pressure monitoring in children and adolescents--our results]. / Kontinuirano mjerenje arterijskog tlaka u djece i adolescenata--nasa iskustva.

UNLABELLED: Objective of the study was to present the results of ambulatory blood pressure monitoring (ABPM) in children and adolescents with hypertension diagnosed by primary care physician. METHODS: we retrospectively reviewed ABPM studies in 76 children. Mean patient age was 14.3 years (4-17 years); 53 boys (69.7%) and 23 girls (30.3%). Children were classified as having either primary or secondary hypertension following a standardised evaluation. According to ABPM data hypertension was defined as mean blood pressure greater than 95 th percentile for age, gender and height and/or blood pressure load (BP load) greater than 25 percent. RESULTS: In 16 (21.1%) children the ABPM studies were normal, leading to a diagnosis of "white coat hypertension" (WCH). Among 50 (65.8%) children with primary hypertension the most (20 or 40% children) had stage 3 hypertension. In secondary hypertension group 6 (60%) of children had stage 3 hypertension. Daytime and nocturnal systolic and diastolic blood pressure values were greater in patients with secondary hypertension compared with patients with primary hypertension. DISCUSSION: The oscillometric monitors for ABPM are generally preferred in children. The high percentage of stage 3 hypertension in both primary and secondary hypertension can be partly explained with normative values used witch were those recommended by consensus group such as the Second Task Force. Daytime and nocturnal systolic and diastolic blood pressure values greater in patients with secondary hypertension correspond to data in literature. CONCLUSIONS: ABPM is important tool in the evaluation and management of childhood hypertension. A normotension in ABPM study will suggest WCH. According toABPM results it is possible to classify hypertension, to identify children who require more detailed evaluation and to asses the efficacy of antihypertensive treatment. The lack of consensus and generaly accepted normative data for pediatric population in ABPM interpretation require further investigation.

Determination of Host Responses to Magnaporthe grisea on Detached Rice Leaves Using a Spot Inoculation Method.

Through the use of standard assays, where conidia of the pathogen Magnaporthe grisea are sprayed onto rice, it is impossible to determine the exact number of conidia in any given area and to predict the locations of disease lesions in the rice blast system. To develop a localized, quantitative inoculation of M. grisea, a novel spot method was investigated. Serially diluted Tween 20 was added to M. grisea conidial suspensions in 0.25% (wt/vol) gelatin to promote adherence of conidia on detached rice leaves. Standard assays indicated no deleterious effects of Tween 20 to rice blast development and 0.02% (vol/vol) Tween 20 was necessary for promoting adherence of spore suspensions to the detached leaves. The spot method was evaluated using three well-characterized races of M. grisea and confirmed with standard assays. Disease reactions of rice to four predominant races of M. grisea were tested concurrently using the spot method and standard assays. Successful application of this assay will help identify novel sources of rice blast resistance and evaluate virulence of M. grisea to aid in breeding resistance to rice blast.

A structural account of substrate and inhibitor specificity differences between two naphthol reductases.

Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 A resolution structure allows for comparisons with the 1.7 A resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH(2) groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

Gain of virulence caused by insertion of a Pot3 transposon in a Magnaporthe grisea avirulence gene.

The avirulence gene AVR-Pita in Magnaporthe grisea prevents the fungus from infecting rice cultivars carrying the disease resistance gene Pi-ta. Insertion of Pot3 transposon into the promoter of AVR-Pita caused the gain of virulence toward Yashiro-mochi, a rice cultivar containing Pi-ta, which demonstrated the ability of Pot3 to move within the M. grisea genome. The appearance of Pot3 in M. grisea seems to predate the diversification of various host-specific forms of the fungus.

Structures of trihydroxynaphthalene reductase-fungicide complexes: implications for structure-based design and catalysis.

BACKGROUND: Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. RESULTS: Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 A (pyroquilon), 2.0 A (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 A (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 in each of the three structures likely serves to stabilize the cationic form of the residue for participation in catalysis. CONCLUSIONS: The pro S hydrogen of NADPH (which is transferred as a hydride to the enzyme's naphthol substrates) is directed toward the carbonyl carbon of the inhibitors that mimic an intermediate along the reaction coordinate. Modeling tetrahydroxynaphthalene and trihydroxynaphthalene in the active site shows steric and electrostatic repulsion between the extra hydroxyl oxygen of the former substrate and the sulfur atom of Met-283 (the C-terminal residue), which accounts, in part, for the 4-fold greater substrate specificity for trihydroxynaphthalene over tetrahydroxynaphthalene.

A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta.

Genetic mapping showed that the rice blast avirulence gene AVR-Pita is tightly linked to a telomere on chromosome 3 in the plant pathogenic fungus Magnaporthe grisea. AVR-Pita corresponds in gene-for-gene fashion to the disease resistance (R) gene Pi-ta. Analysis of spontaneous avr-pita(-) mutants indicated that the gene is located in a telomeric 6.5-kb BglII restriction fragment. Cloning and DNA sequencing led to the identification of a candidate gene with features typical of metalloproteases. This gene is located entirely within the most distal 1.5 kb of the chromosome. When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. Frequent spontaneous loss of AVR-Pita appears to be the result of its telomeric location. Diverse mutations in AVR-Pita, including point mutations, insertions, and deletions, permit the fungus to avoid triggering resistance responses mediated by Pi-ta. A point mutation in the protease consensus sequence abolishes the AVR-Pita avirulence function.

tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.

The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase.

Mutants of Magnaporthe grisea harboring a defective gene for 1,3, 8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6, 8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coli and purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger K(i) for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

Magnaporthe grisea pth11p is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate cues.

Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11-green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues.

Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis.

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.

Crystallization and preliminary x-ray diffraction study of 1 ,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea.

1,3,8-Trihydroxynaphthalene reductase was crystallized in the presence of NADPH and the inhibitor tricyclazole. The crystals are trigonal, space group P3(1)21 or its enantiomorph P3(2)21. Two crystal forms with slightly different cell dimensions were obtained. Form A has unit cell dimensions a = b = 142.6 angstrom, c = 70.1 angstrom and form B cell dimensions a = b = 142.6 angstrom, c = 72.9 angstrom. The diffraction pattern of the latter crystal form extends to 2.5 angstrom resolution.

Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea.

Fungal plant pathogens have evolved diverse mechanisms for penetrating into host plant tissue, ranging from entry through natural plant openings to various mechanisms of direct penetration through the outer surface. The filamentous fungus Magnaporthe grisea can cause disease on many species of the grass (Poaceae) family. The disease on rice, Rice Blast, is of enormous economic importance and biological interest. The mechanism used by this pathogen for breaching the formidable host surface barriers has been studied cytologically and genetically as a model for plant pathology, and represents a remarkably sophisticated achievement of nature. The single-celled appressorium of M. grisea acts as a vessel for the generation and application of perhaps the highest turgor pressures known. The fungus requires and utilizes melanin-derived, osmotically generated pressures estimated at 80 bars to drive an actin-rich cellular protuberance through the surface of a rice leaf or plastic coverslip.

The PWL host specificity gene family in the blast fungus Magnaporthe grisea.

The PWL2 gene, isolated from a Magnaporthe grisea rice pathogen, prevents this fungus from infecting a second host grass, weeping lovegrass. We have investigated the distribution of sequences homologous to PWL2 in M. grisea strains isolated from diverse grass species. Multiple PWL2 homologs with varying degrees of sequence homology were identified. The presence of PWL2 homologs does not correlate with an avirulent phenotype on weeping lovegrass in many cases: some strains were fully pathogenic on weeping lovegrass although they carry multiple PWL2 homologs. Three weakly hybridizing PWL2 homologs were cloned and characterized. One of these, the PWL1 gene previously identified by genetic analysis, functioned to prevent infection of weeping lovegrass. Cloned PWL3 and PWL4 genes were nonfunctional, although PWL4 became functional if its expression was driven by either the PWL1 or the PWL2 promoter. The PWL1, PWL2, and PWL3/PWL4 genes map to different genomic locations. The amino acid sequences of the predicted PWL1, PWL3, and PWL4 proteins have 75, 51, and 57% identity, respectively, to the PWL2 protein. Our studies indicate that PWL genes are members of a dynamic, rapidly evolving gene family.

Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus.

Genetic analysis of host specificity in the rice blast fungus (Magnaporthe grisea) identified a single gene, PWL2 (for Pathogenicity toward Weeping Lovegrass), that exerts a major effect on the ability of this fungus to infect weeping lovegrass (Eragrostis curvula). The allele of the PWL2 gene conferring nonpathogenicity was genetically unstable, with the frequent appearance of spontaneous pathogenic mutants. PWL2 was cloned based on its map position. Large deletions detected in pathogenic mutants guided the gene cloning efforts. Transformants harboring the cloned PWL2 gene lost pathogenicity toward weeping lovegrass but remained fully pathogenic toward other host plants. Thus, the PWL2 host species specificity gene has properties analogous to classical avirulence genes, which function to prevent infection of certain cultivars of a particular host species. The PWL2 gene encodes a glycine-rich, hydrophilic protein (16 kD) with a putative secretion signal sequence. The pathogenic allele segregating in the mapping population, pwl2-2, differed from PWL2 by a single base pair substitution that resulted in a loss of function. The PWL2 locus is highly polymorphic among rice pathogens from diverse geographic locations.

Isolation of the mating-type genes of the phytopathogenic fungus Magnaporthe grisea using genomic subtraction.

Using genomic subtraction, we isolated the mating-type genes (Mat1-1 and Mat1-2) of the rice blast fungus, Magnaporthe grisea. Transformation of M. grisea strains of one mating type with a linearized cosmid clone carrying the opposite mating-type gene resulted in many "dual maters," strains that contain both mating-type genes and successfully mate with Mat1-1 and Mat1-2 testers. Dual maters differed in the frequency of production of perithecia in pure culture. Ascospores isolated from these homothallic crosses were either Mat1-1 or Mat1-2, but there were no dual maters. Most conidia from dual maters also had one or the other of the mating-type genes, but not both. Thus, dual maters appear to lose one of the mating-type genes during vegetative growth. The incidence of self-mating in dual maters appears to depend on the co-occurrence of strains with each mating type in vegetative cultures. In rare transformants, the incoming sequences had replaced the resident mating-type gene. Nearly isogenic pairs produced from three M. grisea laboratory strains were mated to investigate their fertility. One transformant with switched mating type appears to have a mutation that impairs the development of asci when its mating partner has a similar genetic background. The M. grisea Mat1-1 and Mat1-2 genes are idiomorphs approximately 2.5 and 3.5 kb in length, respectively.

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea.

A gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.