Enzyme production by wood-rot and soft-rot fungi cultivated on corn fiber followed by simultaneous saccharification and fermentation

This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.     

The diversity of rhizobia nodulating chickpea (Cicer arietinum) under water deficiency as a source of more efficient inoculants

Chickpea is an important pulse crop grown mainly in the arid and semi-arid regions. The effect of water deficiency on nodulation, biomass production, and competition for nodule occupancy was evaluated in three different soils with two chickpea cultivars, Amdoun I and Chetoui. Two watering regimes were considered; a control that was irrigated three times per week and a water-deficient treatment that was irrigated only one time a week. Results showed that water deficiency significantly decreased the nodule number and the shoot dry weight for both cultivars. Root-nodule bacteria were isolated and characterized by PCR-RFLP of 16S rDNA and nifD-K intergenic spacer. The results show that water deficiency affects the diversity of nodulating rhizobia. The nodulation by Mesorhizobium mediterraneum was reduced while inefficient nodulation by Ensifer meliloti was favoured. In both treatments, chickpea was preferentially nodulated by nifD-K type N6. Analysis for NaCl tolerance showed that most of rhizobia nodulating chickpea under water deficiency are NaCl tolerant. Inoculation with the selected salt-tolerant strain of M. mediterraneum LILM10 increased significantly nodule number and grain yield in the field.     

Virulence spectra and geographical distribution of Mal Secco disease of citrus caused by Phoma tracheiphila in the Mediterranean countries: Tunisia and Italy

This work aimed to find out patterns of virulence variability of a Phoma tracheiphila population of 51 isolates, to determine geographic distribution of Mal Secco disease in citrus orchards of six Mediterranean countries and also to establish correlation between geographic distribution and pathotypic distance of P. tr population structure over our sampling spatial scale. Based on unweighted pair-group method with arithmetic averaging clustering and mean disease rating scores, three distinct virulence groups were identified. The 51 isolates were classified into 20 pathotypes. Extensive virulence variability was detected in 51 isolates of P. tr causing MSD of citrus in the Mediterranean basin. Regression plot between pairwise virulence and geographical distance showed that virulence is independent of the geographical origin and that isolates collected from the same country have different degrees of virulence. The lack of significant correlation between virulence and geographic structure confirmed the absence of isolation-by-distance pattern, suggesting non-regular and non-gradual dispersal of the pathogen over this spatial scale.     

Combining physical, chemical and biological methods for synergistic control of postharvest diseases: A case study of Black Root Rot of carrot

Combining different control methods can improve control efficacy, increase the spectrum of controlled pathogens and reduce the possibility of resistance development. To be successful, however, the different methods need to be compatible: the first treatment should not have any deleterious effect on the succeeding one; preferably, it should contribute to its efficacy. In the last few years, carrot growers in Israel have begun to brush carrots before storage to remove the outer peel of the root. In the present study we show that this practice enhances the appearance of Black Root Rot during storage, a postharvest disease caused by the fungus Thielaviopsis basicola. The chemical fungicide iprodione is usually applied before storage to reduce the development of postharvest diseases. We evaluated the efficacy of combining physical, low-residue chemical and biological control agents as an alternative to the conventional chemical control approach. A technology for the precise application of steam and combined application with stabilized hydrogen peroxide (Tsunami^® 100) or a yeast commercial product (Shemer™) were tested. Used alone, both the steam and Tsunami were highly effective at reducing disease decay but were phytotoxic to the roots. Application of combined treatments of sublethal steam followed by a sublethal dosage of Tsunami or Shemer improved efficacy and disease control by 80 and 86%, respectively. These combinations showed a synergistic effect as compared to each of the treatments alone. The same pattern, effecting up to 54% disease control, was observed with the non-compatible combination of applying Tsunami first, washing it off with water and then applying Shemer. Thus disease-control agents can potentially be used for a short period, then washed off, if needed, and efficiently followed by application of a biological control agent. The biological pathway and mode of action are still under investigation but to the best of our knowledge, this is the first study to mathematically demonstrate synergistic effects of sublethal treatments applied sequentially to control postharvest disease as a potential method to reduce the use of chemicals in fruit and vegetables.     

Development of SCAR markers and a semi-selective medium for the quantification of strains Ach 1-1 and 1113-5, two Aureobasidium pullulans potential biocontrol agents

Aureobasidium pullulans strains Ach 1-1 and 1113-5 are two effective biocontrol agents against Botrytis cinerea and Penicillium expansum on stored apples. In the present work, a monitoring system allowing their identification and quantification was developed. The methodology used consisted of the development of both molecular markers and a semi-selective medium. The random amplified polymorphic DNA (RAPD) technique was applied to a collection of 15 strains of A. pullulans, including Ach 1-1 and 1113-5. Five specific RAPD fragments were amplified for strain Ach 1-1 and three others for strain 1113-5. Among them, a fragment of 528 bp specific to strain Ach 1-1 (generated with the OPR-13 RAPD primer) and another one of 431 bp specific to strain 1113-5 (amplified with the OPQ-03 RAPD primer) were selected, cloned, sequenced, and used to design sequence-characterized amplified region (SCAR) primers. Three different SCAR markers were amplified: two specific to strain Ach 1-1 (189 bp and 387 bp) and one specific to strain 1113-5 (431 bp). These SCAR primers can clearly identify strains Ach 1-1 and 1113-5 among 14 strains of A. pullulans and among eight yeast strains commonly present on apple fruit surfaces. Their selectivity was also tested using DNA extracted from epiphytic microflora of the apple surface. As a semi-selective medium, PDA medium supplemented with 0.5 mg L⁻¹ euparen, 1 mg L⁻¹ sumico, 2.5 mg L⁻¹ hygromycin B, 30 mg L⁻¹ streptomycin sulphate, and 1 mg L⁻¹ cycloheximide was selected. It inhibited the development of the air microflora and appeared highly toxic for the epiphytic microflora of apple surface without altering the growth of the targeted strains Ach 1-1 and 1113-5. The combination of the semi-selective medium and SCAR markers provides a valuable monitoring tool to specifically identify and quantify A. pullulans strain Ach 1-1 and strain 1113-5 and could be used in future studies to evaluate their population dynamics under various laboratory and practical conditions.     

基于高光谱特征和偏最小二乘法的春小麦叶绿素含量估算

叶绿素含量是影响作物生长及产量的主要因素。该研究以2017年6月小型试验田获取的抽穗期春小麦叶绿素含量及其对应的光谱反射率为数据源,对红边(627~780 nm)、黄边(566~589 nm)、蓝边(436~495 nm)、绿边(495~566 nm)、吸收谷和反射峰的最大反射率及反射率总和等16个高光谱特征参数与叶绿素含量之间的相关性进行了分析,并结合偏最小二乘回归法(partial least-squares regression,PLSR)对叶绿素含量进行高光谱建模及验证。结果表明:1)对特定的16个光谱特征参数而言,光谱特征参数绿边最大反射率与春小麦叶绿素质量分数之间的决定系数最低(R~20.5);决定系数较高(R~2≥0.5)的光谱特征参数包括蓝边最大反射率、蓝边反射率总和、黄边最大反射率、黄边反射率总和、红边最大反射率、红边反射率总和、绿边反射率总和、820~940 nm反射率总和及最大反射率、500~670 nm归一化吸收深度和560~760 nm归一化吸收深度,其中820~940 nm反射率总和决定系数达到最高(R~2为0.8);2)利用16个特征参量进行PLSR建模后,发现波段范围在820~940 nm的最大反射率及反射率总和所建立的PLSR估算模型为最优模型,其精度参数R~2p=0.8、RMSEp=2.0 mg/g、RPD=3.2。因此,该模型具有极好的预测能力。该研究为相关研究及当地精准农业提供科学支持和应用参考。     

Differential Responses of Barley Landraces and Improved Barley Cultivars to Nitrogen-Phosphorus Fertilizer

Barley is traditionally grown in the Mediterranean region as a dryland crop, invariably under drought-stressed conditions and often without inputs such as fertilizer. Following research that demonstrated the benefits of fertilization, even under less-than-favourable rainfall condition, fertilizer use on cereals has increased dramatically in the past few decades in countries of West Asia-North Africa. With developments in breeding new barley cultivars for higher yield, combined with disease resistance and environmental adaptability, it is crucial to assess the extent to which such cultivars respond to fertilizer inputs as this may affect aspects of a breeding strategy, particularly the choice of germplasm for adaptation in any particular environment. Thus, we assessed the yield potential of 30 barley cultivars with a range of germplasm types, including new cultivars and landraces, in a greenhouse in two soil types with and without adequate nitrogen and phosphorus fertilizer, i.e., low and high fertility. By comparison with the unfertilized low fertility soils, the fertilizer treatment increased yield parameters by about 10-fold. However, the rankings of some cultivars changed markedly with fertilization; some increased, others showed poor responses and decreased relatively, and two performed well with and without fertilizer. Based on the differential responses at the initial screening stage in the greenhouse, it is possible to identify lines or cultivars that are highly responsive to fertilizers and to incorporate such germplasm for further development to produce high-yielding cultivars for commercial adoption by farmers.     

Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil

In Latin America, rabies cases related to frugivorous bats have been reported since 1930's. Recently, two viruses isolated from Artibeus lituratus were proved to be vampire bat variants by monoclonal antibodies panels [2], but their genetic information is not well known. In this report, four rabies viruses were isolated from frugivorous bats (Artibeus spp.) in Brazil and their nucleoprotein gene sequences were determined. These isolates were found to be genotype 1 of lyssavirus and showed the maximum nucleotide sequence homology of 97.6-99.4% with vampire bat-related viruses in Brazil [6]. These results indicate that the Brazilian frugivorous bat rabies viruses in this study are closely related to vampire bat-related viruses that play a main role in rabies virus transmission to livestock in Brazil.     

Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.