Artificial Inoculum Research Articles

Expression analysis and characterization of resistance (R) genes in contrasting Fusarium wilt resistant chickpea genotypes

Fusarium wilt is one of the most notorious diseases of crop plants. This disease leads to huge crop losses in chickpea with substantial monetary value worldwide. The objective of the current study was to characterise Fusarium oxysporum f. sp. cicris (Foc), the causal agent of chickpea wilt, on morphological basis followed by the assessment of resistance of thirty chickpea germplasm accessions to Foc in a pot experiment. The second objective of this research was to study gene expression and characterisation of specific resistance (R) genes in different Fusarium wilt-resistant chickpea genotypes. The morphological identification and characterization of Foc was done by culturing the fungus potato dextrose agar (PDA) medium. Fusarium wild resistance was assessed by challenging 30 chickpea genotypes to artificial inoculum of Foc by using a pre-defined disease rating scale. Different resistance levels were showed by different chickpea genotypes ranging from highly resistant to highly susceptible. Two accessions demonstrated resistance, 15 were moderately resistant and the rest of the genotypes were moderately susceptible to susceptible. The in-silico expression analysis of R genes showed that two R genes, LOC101499430 and LOC101499568 were upregulated in chickpea roots, indicating their probable role in Fusarium wilt resistance. The genes were further assessed for their expression in response to Fusarium inoculation in contrasting Fusarium wilt resistant and susceptible chickpea genotypes. The resistant genotypes showed significantly higher expression levels of the selected R genes in comparison with susceptible genotypes. This study provides valuable insights into evaluating chickpea germplasm for Fusarium wilt resistance and identification of candidate resistance genes which could be employed for enhancement of Fusarium wilt resistance in chickpea.

Variations Among Crop Seasons and Inoculum Availability Have a Higher Impact on Grapevine Rust Epidemics than Training Systems or Plastic Cover.

Canopy management practices can be effective as part of the integrated management of grapevine diseases. This study aimed to determine whether training systems and plastic covers can contribute to prevent Asian grapevine leaf rust (AGLR) development. Additionally, the influence of crop season and inoculum availability on AGLR development was investigated. Six-season experiments were carried out to characterize 16 epidemics that developed from natural inoculum (NI) or artificial inoculum (AI) sources (NI + AI), conducted in different training systems and with or without the plastic cover. The Richards model was fitted to each AGLR disease progress curve to estimate and compare the onsets and intensities of epidemics using eight curve elements. Principal components analysis (PCA) identified the incidence progress rate, the area under the severity progress curve, final disease severity, time to disease onset, and time to reach the inflection point as the main descriptors for AGLR epidemics. The results showed that AGLR epidemic development was related mainly to differences in inoculum availability and climatic conditions throughout the seasons and to a lower extent to the training system and plastic cover. The earliest disease onset was observed in epidemics when the NI was supplemented with an AI source. Differences in AGRL intensity were correlated to accumulated precipitation, being less severe in autumn-winter than in the spring-summer season. The present findings provided a better understanding of the structure and the seasonal variation of AGLR in cultivar 'Niagara Rosada'. The strategies for reducing and/or delaying inoculum buildup among seasons are discussed.

Identification of resistance germplasm in sesame against root and stem rot (Macrophomina phaseolina) of Sesame

3700 accessions of sesame germplasm were sown during kharif 2021 with susceptible and resistant checks in augmented plot design in 3 meter length row in sick plot (artificial inoculum) condition. Out of these accessions, only 53 accessions were resistant against root and stem rot of sesame and disease incidence was less than 10%.

A reassessment of the fungicidal efficacy of 1,3-dichloropropene, chloropicrin, and metam potassium against Macrophomina phaseolina in strawberry.

The effectiveness of metam potassium, 1,3-dichloropropene, chloropicrin, and different ratios of 1,3-dichloropropene and chloropicrin on the reduction of natural and artificial inoculum of Macrophomina phaseolina were investigated in laboratory and field experiments. Additionally, a multivariate meta-analysis with data from six field trials conducted in Florida from 2012 to 2018 was performed. In small-plot field experiments using drip stakes, the highest rate (468 L ha-1 ) of metam potassium was most effective in controlling M. phaseolina in infected crowns buried at 15.2cm from the point of fumigant injection, whereas none of the rates was able to reduce inoculum buried at 30.5cm. In closed-container experiments, use of the highest rate of 1,3-dichloropropene (168 kg ha-1 ) resulted in the highest level of pathogen control. Different rates of chloropicrin also reduced inoculum when compared to the non-treated control. 1,3-dichloropropene + chloropicrin at different ratios were also highly effective in controlling M. phaseolina. Results from the meta-analysis of open-field experiments indicated that metam potassium and 1,3-dichloropropene + chloropicrin (63:35, v:v) treatments were significantly more effective in reducing M. phaseolina than the 1,3-dichloropropene + chloropicrin (39:60, v:v) treatment; however, metam potassium was not as effective at the side of the beds. 1,3-dichloropropene alone and in mixture with chloropicrin were more effective in reducing inoculum of M. phaseolina than chloropicrin alone, indicating the fungicidal efficacy of 1,3-dichloropropene. Formulation with higher 1,3-dichloropropene concentration performed better than the formulation with higher chloropicrin concentration in field trials. Metam potassium was effective when applied at the highest rate, but with limited lateral movement perpendicular to the drip irrigation line. © 2022 Society of Chemical Industry.

Variations in Pathogenicity of Three Different Forms of Rhizoctonia Inoculum and Assessment of Cultivar Resistance of Sugar Beet

Rhizoctonia solani causes pre-emergence and post-emergence damping-off, as well as crown and root rot of sugar beet (Beta vulgaris L.), which significantly affects the yield returns in the USA and Europe. The pathogen overwinters as sclerotia or melanized mycelium. Traditionally, the resistance of cultivars to R. solani is evaluated by scoring disease reactions at the crowns and roots of older seedlings, thus resistance is not evaluated during seed germination. Moreover, earlier studies evaluated cultivars resistance to R. solani using colonized whole barley or wheat grains which, unlike sclerotia, are artificial inocula of the pathogen that require time, space and technical know-how to produce. Moreover, colonized grains are prone to contamination with other pathogens, consumed by rodents/birds while applied in the field, and are often uneconomic. Considering those limitations, a study was undertaken (1) to develop in vitro methods to generate large-scale sclerotia, (2) to compare pathogenic potentials of sclerotia, mycelia, and colonized barley grains for optimization of dampingoff assays, and (3) to evaluate Rhizoctonia resistance of selected commercial cultivars during the seed germination phase. Comparing six different culture media, we found that R. solani had the highest radial growth (8.9 ± 0.04, cm³) at 8-days and the maximum number of sclerotia produced (203 ± 4.6) at 28-days in CV8 medium. We demonstrated significant differences in pathogenicity of the three different forms of R. solani inocula and susceptibility of cultivars to preand post-emergence damping-off. The highest pre-emergence damping-off and root rot were observed with sclerotia, and the highest post-emergence dampingoff was recorded with both sclerotial and colonized barley inocula. In addition, varietal differences in susceptibility to pre- and post-emergence damping-off were noted. The highest pre-emergence damping-off was recorded on cv Crystal 101RR and lowest in Maribo MA 504. The highest post-emergence damping-off was recorded on BTS 8500 and the lowest in Crystal 467. The maximum mean root rot was observed in BTS 8500, BTS 8606, and Crystal 101R. Our studies demonstrated that sclerotia serve as efficient natural inocula, reemphasized that host-pathogen interactions differ at the early vs. late stages of sugar beet growth, and highlighted the need to reevaluate commercial sugar beet cultivars for resistance at the seed germination stage.

Inheritance and allelic relationship of foliar blast resistance gene(s) in pearl millet

Foliar blast disease in pearl millet (Pennisetum glaucum (L.) R. Br.) caused by Magnaporthe grisea once considered as a minor disease, has become major threat for pearl millet cultivation during recent decade in almost all pearl millet growing areas. The present study is an attempt to understand the inheritance of foliar blast resistance in pearl millet against the natural inoculum of Magnaporthe grisea and also against artificial inoculum of its devastating strain PMg_DI. The experiment was conducted at ICRISAT, Hyderabad and IARI, New Delhi during 2017-19. F2 and backcross populations derived from a susceptible genotype ICMB 95444 and two resistant lines PPMI-10018 and PPMI-2464 were used. Screening of individual plant in F1s, F2s, BCP1s (susceptible parent × F1) and BCP2s (resistant parent × F1) under field and controlled conditions indicated that the resistance is governed by a single dominant gene. The test of allelism was conducted by crossing the resistant parents. No segregation for resistance was observed in F2 generation when screened against the natural inoculum of Magnaporthe grisea and its strain PMg_DI. This indicates that both the resistant parents possess the same gene.

The rust Puccinia arechavaletae, a potential biological control agent of balloon vine (Cardiospermum grandiflorum) in South Africa. II: Host range

Cardiospermum grandiflorum (Balloon vine), a subtropical climber native to America, is rapidly increasing in importance as an invasive environmental weed in South Africa. It is a vigorous climber which invades forest margins and watercourses in sub-tropical areas such as coastal KwaZulu-Natal and Mpumalanga. Robust stems with tendrils enable C. grandiflorum to climb trees and form a dense canopy, completely smothering the underlying indigenous vegetation. Consequently it was targeted for biological control. The microcyclic rust fungus Puccinia arechavaletae was collected from Misiones, Argentina, and established under quarantine conditions in South Africa. Host specificity testing was carried out in the quarantine facilities. A total of 19 plant species were inoculated with the rust fungus, including representatives of 13 genera in the Sapindaceae which are native to southern Africa, three cultivars of the important crop plant Litchi chinensis, and all members of Cardiospermum that occurs in southern Africa. Other than C. grandiflorum, only C. corindum (of South African origin), C. pechuelii (native to Namibia), and C. halicacabum var. halicacabum (introduced weed in South Africa) were susceptible. Occasionally pustules developed on Allophylus natalensis, Dodonaea viscosa and Paullinia pinnata, but these are considered to be the result of the artificial high inoculum loads employed and not representing a susceptible host response. It is necessary to undertake a risk assessment of the potential risk posed to C. corindum and C. pechuelii before P. arechavaletae will be considered for the release as a biocontrol agent against balloon vine in South Africa.

Preservation affects the vegetative growth and fruiting body production of Cordyceps militaris.

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4°C could maintain the fungal vitality and fruiting body producing capacity for at least 12months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.

Characterisation of Phaseolus coccineus interspecific germplasm accessions for disease resistance, grain market class and yield attributes

Sister species of the common bean (Phaseolus vulgaris L.) are an attractive genetic resource to broaden the genetic base of this crop, especially for adaptation to extreme environments. The runner bean (Phaseolus coccineus) in particular, has been shown to contribute to disease resistance and tolerance to low soil fertility, and has been used to introduce these traits into the common bean. The objective of this study was to identify germplasm with agronomic traits suitable for cultivation from an interspecific population (Phaseolus coccineus G35346 x P. vulgaris; SER 16) of 186 bush lines. The lines, coded ALB and one yield check, CAL96, were field evaluated for three rain seasons; 2011a (March-June), 2011b (September-November) and 2012 (March-June) at the National Agricultural Research Laboratories (NARL)-Kawanda, Uganda. Artificial inoculum of bean common mosaic virus (BCMV) was applied to the field experiment in 2011a. The morphological (seed type and growth habit) and agronomic attributes (plant vigour, days to physiological maturity; DPM and days to 50% flowering; DF), reaction to occurring diseases and yield performance were monitored. The lines including root rot resistant and susceptible checks; MLB-49-89A, RWR 719 and CAL 96, were also subjected to inoculum of two major root rot pathogens; Fusarium solani f.sp. phaseoli (isolate FSP3) and Pythium ultimum (isolate MS61) under screen house conditions to select for root rot resistance. Results indicated significant differences (P<0.05) among the interspecific lines for the parameters measured. Days to flowering and to DPM ranged from 31-39 and 81-86, respectively. Field disease pressure was generally low, but in general, 50% of the lines had yield above the mean; while 8% maintained above average yield in all seasons. The superior lines included; ALB169 (mean yield 2,564 kg ha-1), ALB214 (mean yield 2,125 kg ha-1), ALB196 (mean yield 2,084 kg ha-1), ALB5 (2,062 kg ha-1), ALB152 (2,016 kg ha-1), and ALB179 (2011 kg ha-1), compared to the check CAL96 (1,607 kg ha-1). These lines, except ALB169 and ALB179, were resistant root rot. More lines expressed resistance to Fusarium than to Pythium root rot, with 21.5% showing resistance to both root rot isolates. Over 91% of the lines were small or medium seeded (< 35.0 g per 100 seeds), with red monochrome seed pattern; characteristics that are important for farmer acceptance. Key words: Fusarium, Phaseolus coccineus, Phaseolus vulgaris, root rot

Early trans-plasma membrane responses to Tobacco mosaic virus infection

Early trans-plasma membrane behavior after in vivo mechanical inoculation of Nicotiana tabacum with Tobacco mosaic virus (TMV) was investigated and compared to virus quantification in leaf tissues. To identify early events related to virus/host interaction, the systemic virus TMV was used to infect lower leaves and tests were carried out on upper leaves which were not directly infected. Non-invasive microelectrodes were used to estimate trans-plasma membrane electron transport and membrane potential after artificial inoculum of virus, monitoring the plant for the following 15 days. Virus infection was assessed by ELISA and quantified by quantitative RT-PCR. Collected data showed that after 2-day post-inoculation (dpi), TMV was able to modify membrane parameters: transient hyperpolarization of trans-membrane potential was observed until 10 dpi, while redox activity in infected samples was higher compared to control until end of tests. Conversely, ELISA diagnostic test was not able to reveal the virus presence in tobacco leaves until 6 dpi, while leaf symptoms were manifested after 13 dpi.

English

A study was conducted on the induction of coffee wilt disease (CWD) infection with different types of contaminants using a randomized complete block design with four replications. Coffee seedlings were inoculated with infected bark, sub-bark tissues, wood necrotic and an artificial inoculum. Statistical analysis did not show significant differences (P>0.05) between treatments. Results obtained show that all contaminants used induced main symptoms of CWD. Chronologically, wilting appeared an average 55 DAI, followed by leaf browning (60 DAI), defoliation (69 DAI), leaf drying (82 DAI) and seedlings mortality (86 DAI). The lowest rate of wilting and leaf browning (11.1%) was recorded on seedlings inoculated with infected bark, and the highest rate (33.3%) was observed on seedlings inoculated with wood necrotic. Seedlings inoculated with sub-bark tissue expressed 11.1% of leaf drying, and those inoculated with an artificial inoculum presented 22.2% of mortality. Seedlings inoculated with sub-bark tissue expressed 22.1% of defoliation, while those inoculated with artificial inoculum expressed 39.4% of defoliation. The presence of Fusarium xylarioides was confirmed in dead woods of seedlings inoculated with sub-bark tissue, and those inoculated with artificial inoculum. Results obtained confirm the potential danger of wood debris from infected coffee trees, which can act as a source of infection and promote the spread of CWD when dragged through plantations. Key words: Coffea canephora, coffee wilt disease, Fusarium xylarioides, field infection, types of contaminants, Democratic Republic of Congo.

Mid-Infrared (MIR) and Near-Infrared (NIR) Detection of Rhizoctonia solani AG 2-2 IIIB on Barley-Based Artificial Inoculum.

The amount of Rhizoctonia solani in the soil and how much must be present to cause disease in sugar beet (Beta vulgaris L.) is relatively unknown. This is mostly because of the usually low inoculum densities found naturally in soil and the low sensitivity of traditional serial dilution assays. We investigated the usefulness of Fourier transform mid-infrared (MIR) and near-infrared (NIR) spectroscopic properties in identifying the artificial colonization of barley grains with R. solani AG 2-2 IIIB and in detecting R. solani populations in plant tissues and inoculants. The objectives of this study were to compare the ability of traditional plating assays to NIR and MIR spectroscopies to identify R. solani in different-size fractions of colonized ground barley (used as an artificial inoculum) and to differentiate colonized from non-inoculated barley. We found that NIR and MIR spectroscopies were sensitive in resolving different barley particle sizes, with particles that were <0.25 and 0.25-0.5 mm having different spectral properties than coarser particles. Moreover, we found that barley colonized with R. solani had different MIR spectral properties than the non-inoculated samples for the larger fractions (0.5-1.0, 1.0-2.0, and >2.0 mm) of the ground barley. This colonization was confirmed using traditional plating assays. Comparisons with the spectra from pure fungal cultures and non-inoculated barley suggest that the MIR spectrum of colonized barley is different because of the consumption of C substrates by the fungus rather than because of the presence of fungal bands in the spectra of the colonized samples. We found that MIR was better than NIR spectroscopy in differentiating the colonized from the control samples.

Transmission of anthracnose (Colletotrichum lindemuthianum) in dry bean (Phaseolus vulgaris L.) with artificial and natural inoculum in a wet and dry canopy

LeClair, E., Conner, R., Robinson, D. and Gillard, C. L. 2015. Transmission of anthracnose (Colletotrichum lindemuthianum) in dry bean (Phaseolus vulgaris L.) with artificial and natural inoculum in a wet and dry canopy. Can. J. Plant Sci. 95: 913–921. Anthracnose [Colletotrichum lindemuthianum (Sacc. and Magn.) Lams. – Scrib.] is a serious pathogen of dry bean (Phaseolus vulgaris L.). Disease transmission on artificial materials and clothing has been observed in other crops, where equipment and workers transmit pathogens from infected to clean plants. Initial studies in 2008 and 2009 at Exeter, ON, determined that anthracnose transmission in dry bean as measured by resultant disease severity occurred with denim, leather, metal, and rubber using a 107 spores mL−1 prepared artificial spore inoculum in both wet and dry crop canopies. In 2012 and 2013 at Morden, MB, and Ridgetown, ON, the studies were expanded by adding a 105 spores mL−1 prepared artificial and a natural inoculum source. Inoculum source and canopy moisture had the greatest effect on disease severity, while no differences were observed between materials within an inoculum sources. Transmission in wet canopy conditions resulted in a higher infection rate. Canopy moisture impacted the natural inoculum the most. The 107 spores mL−1 inoculum transmitted the most disease followed by natural incidence and 105 spores mL−1 inoculum in wet conditions. In dry conditions 107 spores mL−1 inoculum transmitted the most disease followed by 105 spores mL−1 inoculum and natural incidence. Producers need to recognize that there is real risk for the anthracnose transmission by common materials in dry bean, and take appropriate precautions to prevent it.

Transmission of anthracnose (Colletotrichum lindemuthianum) in dry bean (Phaseolus vulgaris L.) with artificial and natural inoculum in a wet and dry canopy

LeClair, E., Conner, R., Robinson, D. and Gillard, C. L. 2015. Transmission of anthracnose (Colletotrichum lindemuthianum) in dry bean (Phaseolus vulgaris L.) with artificial and natural inoculum in a wet and dry canopy. Can. J. Plant Sci. 95: 913-921. Anthracnose [Colletotrichum lindemuthianum (Sacc. and Magn.) Lams. - Scrib.] is a serious pathogen of dry bean (Phaseolus vulgaris L.). Disease transmission on artificial materials and clothing has been observed in other crops, where equipment and workers transmit pathogens from infected to clean plants. Initial studies in 2008 and 2009 at Exeter, ON, determined that anthracnose transmission in dry bean as measured by resultant disease severity occurred with denim, leather, metal, and rubber using a 107 spores mL-1 prepared artificial spore inoculum in both wet and dry crop canopies. In 2012 and 2013 at Morden, MB, and Ridgetown, ON, the studies were expanded by adding a 105 spores mL-1 prepared artificial and a natural inoculum source. Inoculum source and ...

Review article: Artificial inoculum and inoculation techniques commonly used in the investigation of Fusarium head blight in cereals

Fusarium head blight (FHB) is a disease of small-grain cereals that has a proven negative impact on crop yield, quality and food safety. In this regard, it is one of the most studied diseases of small-grain cereals worldwide. This paper reports the commonly used artificial sources of inoculum and inoculation techniques employed in the study of FHB epidemiology. Spore suspensions and grain spawn are the most popular forms of artificial inoculums. Spray inoculation technique is more commonly used than the point inoculation technique for delivering the inocula to the target sites. Spray inoculation has an advantage over point inoculation in that it can be used to detect both Type I and II resistances to FHB, point inoculation can only detect Type II resistance to FHB. Grain spawn is the commonest soil-surface inoculum used in the study of FHB. Its advantage lies in the fact that it is capable of propagating inoculum over a long period of time compared to other methods. It can also detect the five types of resistances reported (Types I, II, III, IV and V). In order to gain further information regarding the epidemiology of FHB, researchers need to explore other potential sources of artificial inoculum.

First Report of Marasmiellus palmivorus Causing Post-Emergence Damping Off on Coconut Seedlings in Malaysia.

In April and June 2010, coconut seedlings with symptoms of very slow growth, yellowing of leaves, and general abnormal leaf growth were observed in germination beds in Teluk Intan, Perak, Malaysia. The roots were soft, rotten, and brown, extending upward and downward from these lesions. Rhizomorphs and basidiocarps were produced on coconut seeds near the germination eye and identified as Marasmiellus palmivorus according description by Turner (2). Three isolates were obtained by plating surface sterilized symptomatic roots and basidiocarp on malt extract agar (MEA) amended with 85% lactic acid (1 ml added to 11 of the medium). To confirm the identity of the fungus, genomic DNA was extracted from mycelia and basidiocarps of isolates and the large subunit (LSU) region was amplified and sequenced using LR0R/LR7 primers (3). All isolates had identical LSU sequences (GenBank Accession No. JQ654233 to JQ654235). Sequences were identical to each other and 99% similar to a M. palmivorus sequence deposited in the NCBI database (Accession No. AY639434).To confirm pathogenicity, three isolates of M. palmivorus that were obtained from symptomatic plant tissue was inoculated onto seeds of Malaysian Red Dwarf variety. Each isolate was grown in 100 ml of malt extract broth in 250 ml Erlenmeyer flasks and incubated at 27 ± 2°C for 5 days on an orbital shaker (125 rpm). The resulting culture was passed through two layers of sterile cloth. Mycelial suspension was obtained by blending mycelia in 100 ml of sterile water. Seeds were sterilized by soaking in 10% v/v sodium hypochlorite in distilled water for 3 min. The seeds were then rinsed three times over running tap water. The calyx portion of the seed was removed and five holes were made around the germination eye. The seeds were inoculated by injecting 2 ml of suspension into each hole. The control seeds were inoculated with sterile distilled water only. The seeds were transferred to 40-cm diameter plastic pots containing a mixture of sand, soil, and peat in the ratio of 3:2:1, respectively, and steam treated at 100°C for 1.5 h. Pots were placed in the glasshouse with normal exposures to day-night cycles, temperatures of 29 ± 4°C, and high relative humidity (85 to 95%) achieved by spraying water twice daily. After 2 months, 75% of the inoculated seeds failed to germinate. It was speculated that the artificial inoculum was higher than under germination bed conditions. Rhizomorphs and basidiocarps were produced on husk seeds near the germination eye. Seedlings that emerged successfully developed symptoms similar to those observed in the germination bed. No symptoms developed in the noninoculated seeds and seedlings. At 80 days post inoculation, basidiocarps were observed emerging from three diseased seedlings near the germination eye. Three reisolations were made on MEA from root lesions surface sterilized. Pathogenicity tests and LSU sequence analyses indicated that M. palmivorus is the causal agent of the symptoms observed on coconut seedlings. M. palmivorus was first recorded on coconuts and oil palm in the 1920s (1) and attacks the fruit and the petiole on oil palm (2). To our knowledge, this is the first report of M. palmivorus causing post-emergence damping off on coconut seedlings.

Antifungal activity of Meyerozyma guilliermondii: Identification of active compounds synthesized during dough fermentation and their effect on long-term storage of wheat bread

Preliminarily, 146 strains of yeasts were screened for the antifungal activity toward the indicator Penicillium roqueforti DPPMAF1. The strain Meyerozyma guilliermondii LCF1353 was selected and used for dough fermentation. The water/salt soluble extract of the dough was analyzed by HPLC and GC/MS-SPME. The synthesis of the extracellular cell wall-degrading enzyme β-1,3-glucanase and ethyl-acetate was shown. The effect on conidia germination mainly suggested a fungistatic activity. M. guilliermondii LCF1353 was used as starter for dough fermentation in combination with Wickeramomyces anomalus 1695 and Lactobacillus plantarum 1A7, which were previously selected for antifungal activity. The growth of the strains was monitored by plate count and molecular techniques, and competitive or antagonistic interactions among them were excluded. Bread started with the combination of M. guilliermondii LCF1353, W. anomalus LCF1695 and L. plantarum 1A7 showed a more prolonged shelf life compared to the other breads. Fungal growth was delayed at least until 14 days of storage, under conditions of high artificial inoculum. The bread manufactured with the above combination showed good chemical and textural characteristics and, as shown by sensory analysis, it was appreciated for elasticity, color and taste.

Rosellinia necatrix attack according to soil features in the Mediterranean environment

SummaryA study was carried out on Rosellinia necatrix attack on young woody plants, as possibly affected by selected soil features under three water regimes. Six different soil systems representing five agro‐environments and one forest in the Puglia Region (southern Italy – Mediterranean climate) were compared. R. necatrix attack on sweet cherry trees was simulated using artificial inoculum and saplings of Prunus mahaleb, the most widely used rootstock of sweet and sour cherries, monitored during the spring period. Soil features significantly influenced disease score, which did not differ from one water regime to another, even though disease level in the different soils was affected by water content. Rosellinia mahaleb saplings grown in forest soil showed the highest disease score, which differed significantly from that observed in all the agricultural soils tested in this study. Amongst these, disease score was lower in sandy soils than in soils that were richer in loamy fraction.

Mycorrhization of transgenic apple trees with increased resistance against fungal pathogens

The project 'Mycorrhizal symbioses of transgenic apple trees with increased resistance against fungal pathogens' (Subproject of the framework 'Biosafety of transgenic woody plants', supported by the German Federal Ministry for Education and Research) investigates whether the transformation of apple trees with fungal chitinase encoding genes - besides increasing resistance against fungal pathogens - also interferes with mycorrhizal fungi. Pathogenic fungi such as the ascomycete species Venturia inaequalis, the pathogenic agent of apple scab, cause severe damage in apple cultivation. In commercial apple production, pesticides are employed frequently and in high quantities. Alternative methods such as the use of mycoparasitic Trichoderma fungi to control fungal plant pathogens have been explored [1]. Genetic engineering of apple cultivars with Trichoderma chitinases has lead to an increase of the trees’ resistance against apple scab [2]. However, like many fruit tree species, apple trees form symbiotic associations with arbuscular mycorrhizal fungi (AMF) at their roots. AMF receive fixed carbon compounds, while the plants benefit from the symbiosis by enhanced uptake of nutrients and water, promoted growth, and increased resistance against biotic and abiotic stress [3]. Like in most higher fungi, the cell walls of AMF contain chitin as a major skeletal component [4]. Consequently, the symbiosis between apple and AMF may be negatively affected by the expression of Trichoderma chitinase genes within the plant. At the Julius Kuhn-Institute in Dresden, the apple cultivar 'Pinova' was transformed by introducing the construct pBIN (Endo + Nag) into the transgenic lines. With the help of promoter CaMV 35S the transgenic lines constitutively express endochitinase gene ech42 and exochitinase gene nag70 from the biocontrol fungus Trichoderma atroviride. In order to study whether the constitutive expression of ech42 and nag70 has an effect on mycorrhization of plants with transgenic roots (local effect) and – as apple cultivars are propagated vegetatively by grafting– whether expression of ech42 and nag70 in a transgenic scion grafted on a non-transgenic rootstock has an effect on mycorrhization (systemic effect), a series of greenhouse experiments was conducted. In a first experiment, non-grafted clones of wildtype apple cultivar ‘Pinova’ and two transgenic lines were cultivated in artificial substrate and inoculated with two AMF species, Glomus intraradices and G. mosseae. After four months of cultivation, root colonization rates were significantly lower in the transgenic lines compared to the non-transformed cultivar 'Pinova' [5]. Samples were taken again after another four months of cultivation under enhanced inoculation pressure (increased inoculum mass, decreased nutrient availability in the substrate). The experimental approach was broadened by using soil from different field sites in order to supply the sample trees with complex soil matter, AMF adapted to the very soil conditions, and the appropriate soil bacteria which also play a role in the formation of mycorrhizal associations. Two sets of non-grafted sample trees (clones of apple cultivar 'Pinova' and two transgenic lines) were cultivated in the greenhouse; one set in pots with soil from an intensively managed apple plantation, the other set in pots with soil from an extensively managed apple orchard. Root sampling was conducted three and six months after transferring the plants to soil. To assess the systemic effect (expression of ech42 and nag70 in a transgenic scion grafted on a non-transgenic rootstock) on mycorrhization, two further experiments were carried out. Grafted sample trees (clones of apple cultivar 'Pinova' and two transgenic lines, all grafted on commonly grown rootstock M9) were cultivated in the greenhouse in pots with artificial substrate and inoculum of Glomus intraradices and G. mosseae. Root samples were taken after four and eight months. A second set of grafted sample trees was cultivated in pots with soil from an intensively managed plantation in the ‘semi-natural’ environment of a gossamer cage. Root samples were taken after 12 and 24 months. For all experiments, root colonisation rates were determined microscopically. Molecular methods – PCR with taxa specific primer sets [6], cloning of PCR products, Sanger sequencing – were applied to study the impact of the transformation on the composition of the fungal communities associated with the roots of the sample trees cultivated in soil. Chitinase activity in leaves and roots of sample trees was determined using fluorescence spectrometry and substrates 4-methylumbelliferyl N-acetyl-β-D-glucosaminide for exochitinase and 4-methylumbelliferyl β-D-N,N′,N′′-triacetylchitotrioside for endochitinase activity. The results of this biosafety research concerned with potential adverse effects on benefitial soil fungi will be presented and discussed.

DISINFESTATION OF POLYSTYRENE SEEDLING TRAYS FOR SUBSTRATE-BASED FLOAT SYSTEMS IN AUSTRALIA

Internationally, a move by the tobacco industry to substrate-based ‘float systems’ for transplant production has dramatically reduced their use of methyl bromide (MB) for soil disinfestation. The float system utilises expanded polystyrene trays, filled with soil-less substrate, seeded with tobacco, and floated on a common pool of water (containing fertilisers) within polyhouses. Prior to 2005, tobacco growers in Australia (and other countries) disinfested their used trays with MB to reduce the risk of carrying pathogens into the float system. Our research investigated four approaches to provide growers with alternatives to MB for tray disinfestation: (a) improved cultural practices, (b) heat disinfestation, (c) biocidal dips, and (d) alternative fumigants. Results showed there was a strong association between improved hygiene practices by growers (particularly tray handling and storage, p=0.01) and reduced pathogen populations on their used trays. Heat disinfestation with aerated steam (1 h, maximum temperature 80°C) or solarisation (6 wks, maximum temperature 75°C) provided practical and consistent alternatives to MB, reducing fungal populations on used trays to undetectable levels and the viability of artificial inoculum of R. solani embedded in trays to nil. Although biocides (e.g. NaOCl) and fumigants (e.g. methyl iodide) also reduced pathogen populations on trays, they sometimes caused phytotoxicity in transplants. Overall, our research showed that improved hygiene practices and tray disinfestation with steam or solarisation provide viable alternatives to MB for minimising the risk of used trays carrying pathogens into the float system.