Bjorn Welin

Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass

As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.

Pathogen-Induced Accumulation of an Ellagitannin Elicits Plant Defense Response

In an incompatible interaction between Colletotrichum fragariae and strawberry plants, the accumulation of phenolic compounds in plant leaves was observed. A particularly abundant penta-esterified ellagitannin that accumulated in response to pathogen attack was identified as 1-0-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-d-glucopyranose (HeT) by mass spectroscopy and nuclear magnetic resonance. Foliar application of purified HeT prior to inoculation with a virulent pathogen was shown to increase resistance toward C. acutatum in strawberry plants and to Xanthomonas citri subsp. citri in lemon plants. The induced resistance in strawberry was associated with a rapid oxidative burst, callose deposition, a transient increase of salicylic acid in phloem, and induction of gene expression responsive to salicylic acid. Results obtained suggested that HeT could be a common plant defense response molecule capable of inducing pathogen resistance in different plant species.

A Study of the Sugarcane Yellow Leaf Disease in Argentina

Yellow leaf disease, caused by Sugarcane yellow leaf virus (SCYLV), is widespread around the world but very little information is available on this viral disease in Argentina. Therefore, the aims of the study were to assess the presence of SCYLV, analyze its distribution in the main sugarcane production areas of Argentina, characterize the virus, and determine histological alterations caused by its presence. For this purpose, 148 sugarcane samples with and without symptoms were collected in 2011 and 2012 from the province of Tucumán. One additional sample was collected in Salta, a different geographical, agroecological, and producing region. Results showed that SCYLV is widely distributed in commercial varieties of sugarcane throughout Tucumán in both symptomatic and asymptomatic leaves. A low but statistically significant positive correlation with virus detection and disease symptoms was found. BRA-PER was the only genotype detected by reverse-transcription polymerase chain reaction and sequence analysis of the SCYLV capsid protein gene. SCYLV-positive samples showed high starch levels in bundle sheath cells, whereas the asymptomatic ones, probably in an early stage of infection, were found to contain more chloroplasts. Symptomatic noninfected samples presented crystal formation probably associated with phytoplasma infection.

DEVELOPMENT OF PSP1, A BIOSTIMULANT BASED ON THE ELICITOR AsES FOR DISEASE MANAGEMENT IN MONOCOT AND DICOT CROPS

In this work, we present a novel biostimulant for sustainable crop disease management, PSP1, based on the plant defense-elicitor AsES, an extracellular protease produced by the strawberry fungal pathogen Acremonium strictum. Fungal fermentation conditions and downstream processing were determined to maximize extracellular protein production, product stability and a high plant defense-eliciting activity, as monitored by anthracnose resistance in supernatant-treated strawberry plants subsequently infected with a virulent strain of Colletotrichum acutatum. Fermentation batches were shown to reduce anthracnose development by 30–60% as compared to infected non-treated plants. Product formulation was shown to be stable for 6 months when stored at temperatures up to 45°C and toxicological tests showed that PSP1 was harmless to beneficial organisms and non-toxic to mammalian species at concentrations 50 times higher than those used in plant experiments. Furthermore, disease protection studies using dilutions of PSP1 indicated that there is a minimum threshold protease activity needed to induce pathogen defense in strawberry and that this induction effect is dose-independent. A significant characteristic of PSP1 is its broad-range protection against different diseases in various crop species. In soybean, PSP1 reduced the symptomatology by 70% of Corynespora cassiicola, etiological agent of the target spot. This protection effect was similar to the commercial inducer BION 500 WG based on BTH, and both products were shown to induce an oxidative burst and up-regulated PR1-gene expression in soybean. Furthermore, a double PSP1-treatment on greenhouse-grown sugarcane plants provided protection against bacterial red stripe disease caused by Acidovorax avenae and a double foliar application of PSP1 on field-grown wheat plants significantly increased resistance against Fusarium graminearum, causal agent of head blight disease, manifested mainly in an increased seed germination rate. In summary, these disease protection studies demonstrated an effective control against both bacterial and fungal pathogens in both monocot and dicot crop species, which together with its low production cost, effectiveness at low concentrations, long shelf-life, tolerance to high temperatures, harmlessness to non-target organisms and simple handling and application, make PSP1 a very promising candidate for effective and sustainable disease management in many crop species.

Elicitor-Based Biostimulant PSP1 Protects Soybean Against Late Season Diseases in Field Trials

Currently, fungicide application in soybean production accounts for an important amount of global pesticide use, and it is therefore most desirable to find new healthier and more environmental friendly alternatives for the phytosanitary management in this crop. In this study, we present convincing evidence for effective induction of disease protection by the agricultural biostimulant PSP1, a formulation based on the plant-defense eliciting activity of the fungal protease AsES (Acremonium strictum elicitor subtilisin), in multiple field trials in Argentina. PSP1 was shown to combine well with commercial spray adjuvants, an insecticide, a herbicide and fungicides used in Argentinian soybean production without losing any defense-inducing activity, indicating an easy and efficient adaptability to conventional soybean production and disease management in the region. Results from multiple soybean field trials conducted with different elite genotypes at several locations during two consecutive growing seasons, showed that PSP1 is able to induce an enhanced pathogen defense which effectively reduced late season disease (LSD) development in field-grown soybean. This defense response seems to be broad-range as disease development was clearly reduced for at least three different fungi causing LSDs in soybean (Septoria glycines, Cercospora kikuchii and Cercospora sojina). It was noteworthy that application of PSP1 in soybean alone gave a similar protection against fungal diseases as compared to the commercial fungicides included in the field trials and that PSP1 applied together with a fungicide at reproductive stages enhanced disease protection and significantly increased grain yields. PSP1 is the first example of an elicitor-based strategy in order to efficiently control multiple fungal diseases under field conditions in the soybean crop. These results show the feasibility of using induced resistance products as complements or even full-good replacements to currently used chemical pesticides, fulfilling a role as important components of a more sustainable crop disease management system.

The Endophytic Strain Klebsiella michiganensis Kd70 Lacks Pathogenic Island-Like Regions in Its Genome and Is Incapable of Infecting the Urinary Tract in Mice

Klebsiella spp. have been isolated from many different environmental habitats but have mainly been associated with nosocomial acquired diseases in humans. Although there are many recently published sequenced genomes of members of this genus, there are very few studies on whole genome comparisons between clinical and non-clinical isolates, and it is therefore still an open question if a strain found in nature is capable of infecting humans/animals. Klebsiella michiganensis Kd70 was isolated from the intestine of larvae of Diatraea saccharalis but genome analysis revealed multiple genes associated with colonization and growth promotion in plants suggesting an endophytic lifestyle. Kd70 cells labeled with gfp confirmed capability of root colonization and soil application of Kd70 promoted growth in greenhouse grown sugarcane. Further genomic analysis showed that the Kd70 genome harbored fewer mammalian virulence factors and no pathogen island-like regions when compared to clinical isolates of this species, suggesting attenuated animal/human pathogenicity. This postulation was corroborated by in vivo experiments in which it was demonstrated that Kd70 was unable to infect the mouse urinary tract. This is to the best of our knowledge the first experimental example of a member of a pathogenic Klebsiella spp. unable to infect a mammalian organism. A proteomic comparison deduced from the genomic sequence between Kd70 and several other K. michiganensis strains showed a high similarity with isolates from many different environments including clinical strains, and demonstrated the existence of conserved genetic lineages within this species harboring members from different ecological niches and geographical locations. Furthermore, most genetic differences were found to be associated with genomic islands of clinical isolates, suggesting that evolutionary adaptation of animal pathogenicity to a large extent has depended on horizontal gene transfer. In conclusion our results demonstrate the importance of conducting thorough in vivo pathogenicity studies before presupposing animal/human virulence of non-clinical bacterial isolates.