Adult plant resistance in maize to northern leaf spot is a feature of partial loss-of-function alleles of Hm1.

Adult plant resistance (APR) is an enigmatic phenomenon in which resistance genes are ineffective in protecting seedlings from disease but confer robust resistance at maturity. Maize has multiple cases in which genes confer APR to northern leaf spot, a lethal disease caused by Cochliobolus carbonum race 1 (CCR1). The first identified case of APR in maize is encoded by a hypomorphic allele, Hm1A, at the hm1 locus. In contrast, wild-type alleles of hm1 provide complete protection at all developmental stages and in every part of the maize plant. Hm1 encodes an NADPH-dependent reductase, which inactivates HC-toxin, a key virulence effector of CCR1. Cloning and characterization of Hm1A ruled out differential transcription or translation for its APR phenotype and identified an amino acid substitution that reduced HC-toxin reductase (HCTR) activity. The possibility of a causal relationship between the weak nature of Hm1A and its APR phenotype was confirmed by the generation of two new APR alleles of Hm1 by mutagenesis. The HCTRs encoded by these new APR alleles had undergone relatively conservative missense changes that partially reduced their enzymatic activity similar to HM1A. No difference in accumulation of HCTR was observed between adult and juvenile plants, suggesting that the susceptibility of seedlings derives from a greater need for HCTR activity, not reduced accumulation of the gene product. Conditions and treatments that altered the photosynthetic output of the host had a dramatic effect on resistance imparted by the APR alleles, demonstrating a link between the energetic or metabolic status of the host and disease resistance affected by HC-toxin catabolism by the APR alleles of HCTR.

Heterologous expression and antimicrobial activity of OsGASR3 from rice (Oryza sativa L.).

According to an in silico analysis, OsGASR3 (LOC_Os03g55290) from rice (Oryza sativa L.) was predicted to be involved in plant defense mechanisms. A semi-quantitative reverse transcription polymerase chain reaction assay revealed that OsGASR3 is highly expressed in the inflorescences of Thai jasmine rice (O. sativa L. subsp. indica 'KDML 105'). To characterize the biological activity of OsGASR3, we produced an OsGASR3-glutathione S-transferase fusion protein in Escherichia coli Rosetta-gami (DE3) cells for a final purified recombinant OsGASR3 yield of 0.65 mg/L. The purified OsGASR3 inhibited the hyphal growth of Fusarium oxysporum f.sp. cubense and Helminthosporium oryzae at a relatively low concentration (7.5 µg/mL). Furthermore, OsGASR3 exhibited in planta inhibitory activity against Xanthomonas campestris, suggesting its involvement in defense mechanisms, in addition to its previously reported functions affecting growth and development. These observations indicate that recombinant OsGASR3 may be useful for protecting agriculturally important crops against pathogenic microbes.

Direct Estimation of Local pH Change at Infection Sites of Fungi in Potato Tubers.

Fungi can modify the pH in or around the infected site via alkalization or acidification, and pH monitoring may provide valuable information on host-fungus interactions. The objective of the present study was to examine the ability of two fungi, Colletotrichum coccodes and Helminthosporium solani, to modify the pH of potato tubers during artificial inoculation in situ. Both fungi cause blemishes on potato tubers, which downgrades tuber quality and yield. Direct visualization and estimation of pH changes near the inoculation area were achieved using pH indicators and image analysis. The results showed that the pH of the area infected by either fungus increased from potato native pH of approximately 6.0 to 7.4 to 8.0. By performing simple analysis of the images, it was also possible to derive the growth curve of each fungus and estimate the lag phase of the radial growth: 10 days for C. coccodes and 17 days H. solani. In addition, a distinctive halo (an edge area with increased pH) was observed only during the lag phase of H. solani infection. pH modulation is a major factor in pathogen-host interaction and the proposed method offers a simple and rapid way to monitor these changes.

Antioxidant activities and phenolics of fermented Bletilla formosana with eight plant pathogen fungi.

The tubers of Bletilla formosana were fermented with eight plant pathogen fungi, respectively, and antioxidant activities and total phenolic content (TPC) of the crude extracts of fermented products and non-fermented products were investigated. The antioxidant activities were evaluated in three different test systems [DPPH, ABTS radical-scavenging activity, and ferric reducing-antioxidant power (FRAP)]. It was found that the extract of Helminthosporium maydis fermented B. formosana (FBF) possessed the highest TPC and exhibited a significant antioxidant activity compared with non-fermented product and other fermented products. Correlation analysis between antioxidant activities and TPC was also investigated. The good correlation between antioxidant activities and TPC revealed that the phenolic compounds might be the major contributors for the high antioxidant activities of the fermented B. formosana. Two phenolic compounds, curvularin and dehydrocurvularin, were isolated from H. maydis FBF, which had never been reported from plant of orchidaceae or H. maydis. Curvularin exhibited significant antioxidant activities, and was also present at a high concentration (0.373 mg/mg extract sample), implying an important role for the antioxidant activity of H. maydis FBF. This study suggested that proper fermentation processing could improve TPC and antioxidant activities of B. formosana.

Identification and fine mapping of rhm1 locus for resistance to Southern corn leaf blight in maize.

rhm1 is a major recessive disease resistance locus for Southern corn leaf blight (SCLB). To further narrow down its genetic position, F(2) population and BC(1) F(1) population derived from the cross between resistant (H95(rhm) ) and susceptible parents (H95) of maize (Zea mays) were constructed. Using newly developed markers, rhm1 was initially delimited within an interval of 2.5 Mb, and then finally mapped to a 8.56 kb interval between InDel marker IDP961-503 and simple sequence repeat (SSR) marker A194149-1. Three polymorphic markers IDP961-504, IDP B2-3 and A194149-2 were shown to be co-segregated with the rhm1 locus. Sequence analysis of the 8.56 kb DNA fragment revealed that it contained only one putative gene with a predicted amino acid sequence identical to lysine histidine transporter 1 (LHT1). Comparative sequence analysis indicated that the LHT1 in H95(rhm) harbors a 354 bp insertion in its third exon as compared with that of susceptible alleles in B73, H95 and Mo17. The 354 bp insertion resulted in a truncation of the predicted protein of candidate resistance allele (LHT1-H95(rhm) ). Our results strongly suggest LHT1 as the candidate gene for rhm1 against SCLB. The tightly linked molecular markers developed in this study can be directly used for molecular breeding of resistance to Southern corn leaf blight in maize.

Fungi allergens produced by solid-state fermentation process: optimization and allergen characterization.

Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16-160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.

Effects of fly ash and Helminthosporium oryzae on growth and yield of three cultivars of rice.

A 120-day greenhouse experiment was conducted to study the effects of various fly ash concentrations (0%, 20%, 40%, 60%, 80% and 100% vol/vol) with normal field soil and Helminthosporium oryzae on the growth and yield of three cultivars (Pusa Basmati, Pant-4 and Pant-10) of rice, Oryza sativa L. Application of 20% and 40% fly ash with soil caused a significant increase in plant growth and yield of all the three cultivars. Forty percent fly ash caused a higher increase in growth and yield than did 20%. Sixty percent, 80% and 100% fly ash had an adverse effect on growth and yield of all the three cultivars, the maximum being with 100% fly ash. Inoculation of H. oryzae had an adverse effect on the growth and yield, Pant-10 suffered higher damage by H. oryzae than Pusa Basmati and Pant-4. Pant-10 also exhibited higher infected leaf area and greater disease symptoms of H. oryzae than did Pusa Basmati and Pant-4. Plants grown in 100% fly ash suffered higher reductions in growth and yield with H. oryzae than plants grown in pure soil or in 20% or 40% fly ash. In general, plant growth was best in Pusa Basmati followed by Pant-4 and Pant-10, while yield was higher in Pant-4 followed by Pant-10 and Pusa Basmati.

Molecular size and net charge of pathogenesis-related enzymes from barley (Hordeum vulgare L., v. Karat) infected with Drechslera teres f. teres (Sacch.) Shoem.

Molecular size and net charge of isoforms of pathogenesis-related (PR) chitinase, beta-1,3-glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., v. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time-dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL-MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703-709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxidases increased considerably after infection with Drechslera teres. The molecular masses of peroxidases 1 and 3 were estimated to be 38 +/- 5 and 42 +/- 7 kDa and their apparent valences at pH 8.4 were Z = 3.13 and 3.20, respectively. Amongst the chitinase isoforms, chitinase 1 and chitinase 2 appeared after infection, while chitinase 3 was also observed in uninfected leaves of barley. The molecular mass of chitinase 3 (31 +/- 6 kDa; f/fo = 1.20) was larger than that of chitinase 1 (20 +/- 2 kDa; f/fo = 1.04) and chitinase 2 (23 +/- 3 kDa; f/fo = 1.06). The valence of constitutive chitinase 3 (Z = 1.44 +/- 0.81) at pH 8.4 was lower than that of adaptive chitinase 1 (Z = 3.27 +/- 1.02) and chitinase 2 (Z = 2.96 +/- 1.38). Infection of barley leaves with Drechslera teres also induced the hydrolytic enzyme beta-1,3-glucanase 1; beta-1,3-glucanase 2 appeared in uninfected and in infected leaves. Constitutive beta-1,3-glucanase 2 was smaller (molecular mass 19 +/- kDa; f/fo = 1.05) than adaptive beta-1,3-glucanase 1 (molecular mass 26 +/- 4 kDa; f/fo = 1.07). The valence of adaptive beta-1,3-glucanase 1 (Z = 9.58 +/- 4.17) was approximately threefold that of beta-1,3-glucanase 2 (Z = 2.80 +/- 0.93).