Non-inoculated Control Research Articles

First Report of Alternaria japonica, a causal agent of black spot, on kale in South Carolina, United States.

In January 2020, charcoal gray, dull lesions were observed on leaves of organic kale (Brassica oleracea var. acephala) cv. Darkibor in two fields in Lexington County, South Carolina, the county with the most acres of leafy brassicas in the state. Leaf spots, also visible on the leaf underside, covered <5% of the leaf area. No spores were present. Portions of leaf spots from eight leaves, four per field, were cultured on one-quarter-strength potato dextrose agar (PDA/4). Eleven isolates of Alternaria spp. were recovered. Isolates ALT12 and UL3 were cultured in A. solani medium and DNA was extracted (Maiero et al. 1991). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1), RNA polymerase second largest subunit (rpb2), and Alternaria major allergen (Alt a 1) genes were amplified with the primer pairs V9G/ITS4, EF1-728F/EF1-986R, RPB2-5F2/FRPB2-7cR, and Alt-for/Alt-rev, respectively, and sequenced (Woudenberg et al. 2014). Sequences for isolates ALT12 and UL3, collected from different leaves in the same field, were identical to each other and to isolate AC97 (ITS accession number: LC440597; tef1: LC482018; rpb2: LC476803; Alt a 1: LC481628) of A. japonica Yoshii (Nishikawa and Nakashima 2020). ITS, tef1, repb2, and Alta a 1 sequences for each isolate were deposited in GenBank under the accessions MW374952, MW389653, MW389655, and MW389657 for ALT12 and MW374951, MW389652, MW389654, and MW389656 for UL3, respectively. Conidia of A. japonica (20 of ALT12, 10 of UL3) produced by 7-day-old cultures on Spezieller Nährstoffarmer Agar measured 62.1 ± 11.4 x 18.8 ± 2.2 μm (standard deviation). Median numbers of transverse and longitudinal septae were 6 (4 to 8) and 2 (1 to 3), respectively. Conidia formed singly or in chains of two. Cells were constricted around the transverse septae (Nishikawa and Nakashima 2020; Woudenburg et al. 2014). Chlamydospores were present in cultures of ALT12. ALT12 was pathogenic on kale cv. Darkibor and Winterbor inoculated in a greenhouse following procedures of Al-Lami et al. (2019). Four replicate pots with two plants each were used; plants were 6, 9, and 5 weeks old in trials 1, 2, and 3, respectively. The oldest three leaves of each plant were spray inoculated with a suspension of 5 x 105 conidia/ml; noninoculated control plants were sprayed with water. All plants were kept for 48 h at 100% RH, then moved to a bench in a greenhouse held at 21/16°C day/night temperatures. The second and third oldest leaves were rated 13 days after inoculation. Small gray or black spots developed on inoculated leaves and petioles in all trials, and on one noninoculated leaf in trial one. Disease incidence on inoculated leaves (73.1%) was greater than on noninoculated leaves (0.05%) (P<0.0001). Cultivars did not differ in susceptibility (P=0.12). Portions of lesions on inoculated leaves and portions of noninoculated leaves were cultured onto PDA/4 amended with antibiotics (Keinath 2013). A. japonica was reisolated from 46 of 50 inoculated leaf blades; 22 of 28 inoculated petioles; and 1 of 8, 0 of 8, and 0 of 7 noninoculated leaves in the three trials, respectively. Growers in South Carolina consider black spot, or Alternaria leaf spot, the most important fungal disease on organic kale. The presence of a second causal agent in addition to A. brassicae may increase disease occurrence. A. japonica previously was reported on arugula in California (Tidwell et al. 2014). This is the first report of A. japonica in the eastern United States.

English

Rotten fruits are known to cause major losses in post-harvest conservation. One of the causal pathogen of pineapple rot was investigated and the antifungal effects of selected plants extracts were evaluated in vitro against fungal pathogen; Pestalotiopsis microspora. The fruits samples were randomly collected from farms after harvest. The samples were collected in Awae; the experiment was conducted at Agricultural Research Institute for Development located in Yaounde, Cameroon. The fruits samples were inoculated on potato dextrose agar and pure culture of fungal pathogen responsible for pineapple rot obtained. The pathogen was isolated, and identified on the basis of morphological features. The pathogenicity test was conducted to determine that the fungus is responsible for the rot symptoms. The efficacies of three plant species, namely: Allium sativum, Syzygium aromaticum and Zingiber officinale were tested in vitro at concentrations 20, 40 and 80% on mycelial growth inhibition of the causal agent. Aqueous and ethanolic extracts for concentrations were used. Distilled water and the fungicide Mancozeb 80WP (800 g/kg) were used as negative and positive controls, respectively. The pathogenicity test confirmed that P. microspora fungus is responsible for pineapple fruit rotting. The non-inoculated controls showed no symptoms of fruit rot. Results of antifungal tests showed that after 08 days, aqueous and ethanolic extracts of A. sativum and S. aromaticum were the most effective. The mycelial growth inhibition was total with aqueous and ethanolic extracts of A. sativum for concentrations 20, 40 and 80%. Total inhibition was also recorded with ethanolic extracts of S. aromaticum for all the tested concentrations.&nbsp; The overall result of this study reveals that aqueous and ethanolic extracts of A. sativum and S. aromaticum can be used to control P. microspora as they completely inhibited the growth of the pathogen that can contribute in post-harvest conservation of pineapple fruits. Key words: Ananas comosus, antifungal activities, extracts, pathogenicity test, Pestalotiopsis microspora.

The Effect of Controlled-Release Carvacrol on Safety and Quality of Blueberries Stored in Perforated Packaging.

The objective of this research was to evaluate the use of a controlled-release carvacrol powder to delay storage decay and maintain the safety of blueberries. The controlled-release carvacrol powder was a microcapsule of carvacrol (11% (w/w) active carvacrol) surrounded by a pectin/sodium alginate matrix. The microcapsules were packed in an air-permeable pouch, and then attached to the top of a clamshell filled with blueberries. The blueberries, inoculated with Escherichia coli or Colletotrichum acutatum, or non-inoculated control, were monitored for microbial growth and quality for 10 days at 10 °C and 5 days at 20 °C. Three treatments were compared: controlled-release microencapsulated carvacrol, non-encapsulated carvacrol, and control. The results showed that both the microencapsulated carvacrol and the non-encapsulated carvacrol treatments significantly reduced the populations of yeast and mold, and of E. coli and mesophilic aerobic bacteria. The microencapsulated carvacrol treated berries retained better quality due to significantly lower weight loss than control after 10 days at 10 °C. Sensory panelists found that the microencapsulated carvacrol berries had significantly higher overall blueberry flavor and lower discernible off-flavor in comparison with the non-encapsulated treatment after 3 days at 20 °C. The fruit internal quality, including total soluble solids content (SSC), and titratable acidity (TA), was not significantly affected by any treatment. These results indicate that pectin/sodium alginate controlled-release microencapsulated carvacrol can be used for the preservation of blueberries or other small fruit.

Typhlitis induced by Histomonas meleagridis affects relative but not the absolute Escherichia coli counts and invasion in the gut in turkeys

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcusalactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

Enhancement of Faba Bean (Vicia faba L.) Yield Using Elite Rhizobial Inoculants on Acidic Soils of the Central Highlands of Ethiopia

This study was intended to evaluate the inoculation response of faba bean to six indigenous rhizobial isolates, 100 kg ha-1 DAP as a positive control and non-inoculated negative control under acidic field conditions at Damotu (in 2019) and Chiri (in 2020) of Ejere district, Ethiopia. The experiments were laid out in a randomized complete block design (RCBD) with three replications and a plot size of 4 m x 3 m. The result confirmed the existence of a significant difference between treatments (p ≤ 0.05) in aboveground biomass, grain, and haulms yields. The highest grain yields (3378.3 kg ha-1 and 3286.7 kg ha-1) were obtained from inoculation of rhizobial isolate FB-EM-11 at both experimental sites. The second higher grain yields were obtained from inoculation of rhizobial isolates FB-NS-03 (3376.7 kg ha-1) at Damotu and FB-EM-07 (3269.5 kg ha-1) at Chiri. Based on the two successive years’ grain yield response, FB-EM-11(3332.6 kg ha-1) and FB-NS-03(3198 kg ha-1) were the first and the second most performing isolates at Ejere district. Moreover, from the partial budget analysis result, FB-EM-11, FB-NS-03, and FB-EM-07 were the most promising isolates that showed the higher marginal rate of return 8309%, 7344%, and 6865%, respectively. Thus, these rhizobial isolates are the best promising candidate for further verification over the farmers’ field at different agro-ecologies to identify candidates for the development of commercial faba bean rhizobial inoculants in acid-prone faba bean growing areas of Ethiopia.

Mitigation of Salt Stress by Mycorrhizal Inoculation on Nitraria schoberi as a Native Landscape Plant in the Arid regions

Increasing the salinity in the water and soil can negatively affect plant growth and development. Mycorrhizal fungi application is one of the ways to reduce the undesirable effect of salt stress on plants. An experiment was conducted in 2017 to assess the effect of salt stress on Nitraria schuberi, as a native Iranian plant in arid regions, inoculated with mycorrhizal fungi. Seedlings of this plant were treated under three different levels of NaCl in three stages. The stages including low salt concentrations (0, 20, 60, and 100 mM NaCl), medium salt concentrations (0, 40, 120, and 200 mM NaCl) and high salt concentrations (0, 80, 240, and 400 mM NaCl). Mycorrhizal treatment including two levels: non-inoculated (control) and mycorrhizal inoculated. Experimental designs were factorials (4×2 treatments) based on the completely randomized design with four replications. In this study, the content of chlorophyll, carotenoid, sugar, proline and Na, Mg, K, Fe and Ca were measured. The results indicated that with increasing salinity levels from the first (low) to third (high) stage, chlorophyll content was decreased while carotenoid, proline, and sugar were increased. The application of NaCl salinity led to a reduction in Fe and enhancement in Na. In the mycorrhizal plants, sugar content decreased but magnesium, calcium and potassium levels increased. Based on these findings it seems that Nitraria schuberi is a salt tolerant plant and mycorrhizal fungi can mitigate salinity stress in this plant. Therefore this plant could be applied in the urban landscape of arid and semi-arid regions.

Productivity Improvement of Faba Bean (Vicia faba L.) through Elite Rhizobial Inoculants in the Central Highlands of Ethiopia

This study envisioned to assess the inoculation response of faba bean to six indigenous rhizobial isolates, 100 kg ha-1 DAP as a positive control and non-inoculated negative control under farmers’ field conditions at Welmera district, Ethiopia. The results revealed the presence of significantly different treatments (p ≤ 0.05) in grain yield. The highest grain yields (3966 kg ha-1and 3694 kg ha-1) were obtained from inoculation of rhizobial isolates (FB-AR-13 and FB-1018) during the 2019/20 and 2020/21 cropping seasons. Based on the two successive year’s average grain yield response, FB-AR-13 (3966 kg ha-1) became the first most performing isolate. The partial budget analysis results also showed that FB-AR-13 was the most promising isolate that showed a higher MRR 6422 %, which is 43% higher than the MRR of the local standard check FB-1018. Therefore, isolate FB-AR-13 is the best promising candidate for the development of commercial faba bean rhizobial inoculant in the central high lands of Ethiopia after further verification over different farmers' fields at different agro-ecologies.

Enteric permeability and inflammation associated with day of hatch Enterobacteriaceae inoculation

Early exposure to Enterobacteriaceae may result in inappropriate microbial colonization of the gastrointestinal (GI) tract, induce mild GI inflammation, alter immune system development, and predispose poultry to opportunistic infection. Four experiments were conducted to test Enterobacteriaceae isolates Escherichia coli LG strain (LG), E. coli Huff strain (Huff), Salmonella Enteritidis LB (SE) and Salmonella Typhimurium (ST) on ability to induce GI inflammation. All 4 experiments included a noninoculated control, and day of hatch (DOH) oral inoculation of LG, Huff, SE and ST in experiment 1, LG and SE in experiment 2, and LG, Huff, SE, and ST in experiment 3. Experiment 4 included LG, Huff, a noninoculated control (NIC), and Clostridium perfringens only (NCP) wherein birds received oral C. perfringens challenge on d15-16 to induce necrotic enteritis. Body weight was measured, yolk sacs and spleens were collected, and blood was obtained for serum fluorescein isothiocyanate dextran (FITC-d) recovery and alpha-1-acid glycoprotein (A1GP) concentrations. Samples were taken weekly through 2 wk of age in experiments 1 and 2, or 4 wk of age in experiments 3 and 4. Increased FITC-d recovery was observed for LG and SE on d13 in experiment 2 (P < 0.05), and C. perfringens only birds on d27 in experiment 4 (P < 0.05) as compared to noninoculated controls. Each experiment resulted in notable differences in A1GP serum concentrations over time, with fluctuations in A1GP patterns through d14 based on DOH inoculation (P < 0.05). Over time, A1GP was increased for DOH inoculated birds from d 22 to 29, the fourth wk of life, and d 2-29, the entire experiment, vs. noninoculated controls in experiment 3 (P < 0.05). Similarly, NCP and LGCP showed increased A1GP from d 20 to 27 and d 6 to 27, vs. NIC in experiment 4 (P < 0.05). In experiment 4, C. perfringens challenge resulted in earlier A1GP response in DOH inoculated birds, d 17-20, as compared to NCP birds, d 20-27 (P < 0.05). These results suggest early Enterobacteriaceae exposure may influence early inflammatory state in the GI tract and may also alter patterns of inflammation and responsiveness to pathogens.

Combining microorganisms in inoculants is agronomically important but industrially challenging: case study of a composite inoculant containing Bradyrhizobium and Azospirillum for the soybean crop

The increasing global perception of the importance of microbial inoculants to promote productivity and sustainability in agriculture prompts the adoption of bio-inputs by the farmers. The utilization of selected elite strains of nitrogen-fixing and other plant-growth promoting microorganisms in single inoculants creates a promising market for composite inoculants. However, combining microorganisms with different physiological and nutritional needs requires biotechnological development. We report the development of a composite inoculant containing Bradyrhizobium diazoefficiens and Azospirillum brasilense for the soybean crop. Evaluation of use of carbon sources indicates differences between the microbial species, with Bradyrhizobium growing better with mannitol and glycerol, and Azospirillum with malic acid and maleic acid, allowing the design of a formulation for co-culture. Species also differ in their growth rates, and the best performance of both microorganisms occurred when Azospirillum was inoculated on the third day of growth of Bradyrhizobium. The composite inoculant developed was evaluated in five field trials performed in Brazil, including areas without and with naturalized populations of Bradyrhizobium. The composite inoculant resulted in symbiotic performance comparable to the application of the two microorganisms separately. In comparison to the single inoculation with Bradyrhizobium, co-inoculation resulted in average increases of 14.7% in grain yield and 16.4% in total N accumulated in the grains. The performance of the composite inoculant was similar or greater than that of the non-inoculated control receiving a high dose of N-fertilizer, indicating the importance of the development and validation of inoculants carrying multiple beneficial microorganisms.

Halotolerant Bacillus spizizenii FMH45 promoting growth, physiological, and antioxidant parameters of tomato plants exposed to salt stress.

Bacillus spizizenii is for the first time described as a plant growth salt-tolerant bacterium able to alleviate salt stress in crop plants by improving physiological parameters and antioxidant defense mechanisms. Agricultural soil salinization is a serious issue worldwide affecting agricultural yield. Plant growth promoting bacteria can enhance salt tolerance and plant yield. Bacillus spizizenii FMH45 has been shown to inhibit fungal attacks in tomato fruits and to augment tomato seed germination in presence of abiotic stresses. During this study, we reported for the first time B. spizizenii as a salt-tolerant bacterium able to alleviate salt stress in tomato plants. B. spizizenii FMH45 was examined in vitro for its potential to produce several plant growth promoting characters (siderophores, IAA, and phosphate solubilization) and hydrolytic enzymes (cellulase, glucanase and protease) in the presence of saline conditions. FMH45 was also investigated in vivo in pot experiments to evaluate its ability to promote tomato plant growth under salt stress condition. FMH45 inoculation, enhanced tomato seedling length, vigor index, and plant fresh and dry weights when compared to the non-inoculated controls exposed and not exposed to a regular irrigation with salt solutions containing: 0; 3.5; 7; and 10g L-1 of NaCl. FMH45-treated plants also presented improved chlorophyll content, membrane integrity (MI), and phenol peroxidase (POX) concentrations, as well as reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels under saline conditions with a significant salinity × strain interaction. Furthermore, FMH45 inoculation significantly decreased endogenous Na+ accumulation, increased K+ and Ca2+ uptake, and thereby improved K+/Na+ and Ca2+/Na+ ratios. This study proves that bio-inoculation of FMH45 efficiently increases salt tolerance in tomato plants. This sustainable approach can be applied to other stressed plant species in affected soils.

Effects of Hybrid Susceptibility and Inoculation Timing on Goss's Bacterial Wilt and Leaf Blight Severity and Corn Yield.

Goss's bacterial wilt and leaf blight (Goss's wilt) of corn is the most important corn disease in North Dakota (ND), and yield loss due to the disease has not been reliably quantified in northern corn growing regions. To help quantify the amount of yield loss caused by Goss's wilt, a total of six field experiments were conducted from 2015 to 2017. Experiments were designed in a randomized complete block with a split plot arrangement. Hybrids served as main plots and Clavibacter nebraskensis inoculation timings as subplots. Three hybrids were used and classified as susceptible, moderately susceptible, and resistant. Inoculation timings included a noninoculated control, six to 10 leaf collars (V6 to V10), reproductive silk stage (R1), or a sequential combination of V6 to V10 and R1. A high level of disease (greater than 50% on susceptible hybrid) occurred in three experiments, a low level of disease (less than 5% on susceptible hybrid) in one experiment, and no disease was reported in two experiments. A combined analysis of the high disease experiments indicated yield losses of 34 to 41% on the susceptible hybrid when C. nebraskensis inoculation occurred at V6 to V10. Yield losses of 22 to 25% occurred on the moderately susceptible hybrid when C. nebraskensis inoculation occurred at V6 to V10, and statistical differences in yield loss were not found among inoculations timings on the resistant hybrid. Correlation analyses suggest that for every 1% increase in R1 disease severity on the susceptible hybrid, yield was reduced by 117 kg/ha (1.9 bu/acre). The current study further demonstrates the importance of hybrid resistance and provides updated yield loss information on Goss's wilt in a northern corn growing region.

The Endophytic Fungus Piriformospora indica Reprograms Banana to Cold Resistance.

Banana (Musa spp.), one of the most important fruits worldwide, is generally cold sensitive. In this study, by using the cold-sensitive banana variety Tianbaojiao (Musa acuminate) as the study material, we investigated the effects of Piriformospora indica on banana cold resistance. Seedlings with and without fungus colonization were subjected to 4 °C cold treatment. The changes in plant phenotypes, some physiological and biochemical parameters, chlorophyll fluorescence parameters, and the expression of eight cold-responsive genes in banana leaves before and after cold treatment were measured. Results demonstrated that P. indica colonization reduced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) but increased the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of soluble sugar (SS) and proline. Noteworthily, the CAT activity and SS content in the leaves of P. indica-colonized banana were significant (p < 0.05). After 24 h cold treatment, the decline in maximum photochemistry efficiency of photosystem II (Fv/Fm), photochemical quenching coefficient (qP), efficient quantum yield [Y(II)], and photosynthetic electron transport rate (ETR) in the leaves of P. indica-colonized banana was found to be lower than in the non-inoculated controls (p < 0.05). Moreover, although the difference was not significant, P. indica colonization increased the photochemical conversion efficiency and electron transport rate and alleviated the damage to the photosynthetic reaction center of banana leaves under cold treatment to some extent. Additionally, the expression of the most cold-responsive genes in banana leaves was significantly induced by P. indica during cold stress (p < 0.05). It was concluded that P. indica confers banana with enhanced cold resistance by stimulating antioxidant capacity, SS accumulation, and the expression of cold-responsive genes in leaves. The results obtained from this study are helpful for understanding the P. indica-induced cold resistance in banana.

Diaporthe Seed Decay of Soybean [Glycine max (L.) Merr.] Is Endemic in the United States, But New Fungi Are Involved.

Diaporthe seed decay can compromise seed quality in soybean [Glycine max (L.) Merr.] in the warm and humid production areas of the United States during crop maturation. In the current study, 45 isolates of Diaporthe were recovered from seed sampled from soybean fields affected by Diaporthe-associated diseases in eight U.S. states in 2017. The isolates obtained belonged to 10 species of Diaporthe based on morphology and phylogenetic analyses of the internal transcribed spacer, partial translation elongation factor 1-α, and β-tubulin gene sequences. The associated species included D. aspalathi, D. caulivora, D. kongii, D. longicolla, D. sojae, D. ueckerae, D. unshiuensis, and three novel fungi, D. bacilloides, D. flavescens, and D. insulistroma. One isolate each of the 10 species was examined for pathogenicity on seed of cultivar Sava under controlled conditions. Seven days postinoculation, significant differences in the percentages of decayed seeds and seedling necrosis were observed among the isolates and the noninoculated control (P < 0.0001). While the isolates of D. bacilloides, D. longicolla, and D. ueckerae caused a significantly greater percentage of decayed seeds (P < 0.0001), the isolate of D. aspalathi caused the greatest seedling necrosis (P < 0.0001). The observation of new fungi causing Diaporthe seed decay suggests the need for a more comprehensive survey in U.S. soybean producing areas since members of the genus Diaporthe appear to form a complex that causes seed decay.

Growth enhancement of the highly prized tropical trees siamese rosewood and burma padauk

This study aims to evaluate the efficiency of the co-inoculation of arbuscular mycorrhizal fungi (AMF) and mycorrhizal helper bacteria (MHB) to promote the growth of siamese rosewood and burma padauk seedlings. The experiment was carried out in the pot experiment for 120 days. Three different species of AMF, Glomus clarum KKU−CLD−1, Clomus proliferum PC2−2 and Acaulospora mellea KKU−NBP−NB−2, were individually inoculated with and without the Stenotrophomonas maltophilia KKU−CLD−4.3−3−1. The results showed that the mycorrhizal helper bacteria strain S. maltophilia KKU−CLD−4.3−3−1 isolated from C. proliferum PC2−2 possessed high plant-growth promoting properties. The co-inoculation of our AMF species and MHB, especially in the treatment of C. proliferum PC2−2 and S. maltophilia KKU−CLD−4.3−3−1 could provide higher plant biomass, more nitrogen and phosphorus uptake and higher chlorophyll A, B contents compared to the non-inoculated control. The maximum growth of both types of seedlings was promoted by the treatment consisting of A. mellea KKU−NBP−SB−2, S. maltophilia KKU−CLD−4.3−3−1, and rock phosphate fertilizer. Co-inoculation of AMF and MHB could better promote plant growth parameters and improved phosphorus and nitrogen uptake in both types of seedlings than in the treatment with single inoculation. These suggested that our AMF and MHB were capable of being used as biofertilizer for the growth of Siamese rosewood and Burma padauk seedlings.

First Report of Fusarium venenatum causing foot and root rot of wheat (Triticum aestivum) in Germany.

Field experiments were established in the 2018/19 and 2019/20 growing seasons at the experimental station in Neu-Eichenberg, Hessen, Germany to examine the suitability of multiple wheat cultivars for intercropping with pea and the effect of mixtures on diseases. Approximately 600 wheat tillers (BBCH 49-61, depending on the year and cultivar) were sampled in each year and assessed for severity of foot (lower stem) rot symptoms. Fungi from plants with Fusarium-like symptoms exhibiting reddish-brown discolorations on the stems were isolated following the methods described in Šišić et al. (2018). Surface disinfected (3% NaOCl for 10s) wheat stems and roots were cut into three 1 cm long pieces and placed on COONS agar (Coons, 1916). Following 7 - 12 days of incubation under constant blacklight blue fluorescent light, pure cultures were generated on potato dextrose and synthetic nutrient-poor agar (Nirenberg, 1976) using the hyphal tip transfer technique, and the resulting colonies examined microscopically. Based on morphology ca 15% of all Fusarium isolates recovered belonged to a distinct taxonomic unit and were initially identified as F. sambucinum-like (Leslie and Summerell, 2006). The identity of 16 randomly selected isolates (GenBank accession numbers MW085924 - MW085939) was confirmed by sequencing a portion of the translation elongation factor 1-alpha gene region (O'Donnell et al., 1998). Blast analysis in the FUSARIUM-ID (Geiser et al., 2004) and the NCBI databases revealed >99 to 100% identity match with the Fusarium venenatum accession numbers NRRL 22196, FRC R-09186 and MRC 2394. Pathogenicity tests were conducted on wheat cultivar Torborzo using six F. venenatum isolates. Inoculum was prepared using a sterile sand-millet mix infested with six agar plugs of each of the isolates. Once fully colonized, the inoculum was mixed with sterilized sand in a 1:7 ratio (by volume), transferred to 300 ml pots and 4 surface sterilized wheat seeds (5 min 70% alcohol) were sown in each pot. Non-inoculated controls were amended with sterilized inoculum. The experiment was conducted in a greenhouse in a completely randomized design with 5 replicates. Disease symptoms were assessed after four weeks. All isolates induced reddish-brown discolorations on the lower stems similar to those observed in the field grown plants. In addition, the infected plants developed dark brown to black discolorations on the crowns and roots and showed clear signs of stunted root growth. These symptoms were further accompanied by chlorosis (yellowing) of the lower leaves starting from the leaf tip. All isolates were successfully reisolated from the infected wheat plants but not from the controls. To the best of our knowledge, this is the first report of F. venenatum causing foot and root rot of wheat in Germany. Results from field and greenhouse inoculation experiments indicate that F. venenatum may be an important pathogen of wheat in Germany. Further studies on distribution and relative abundance of the species in the Fusarium foot and root rot complex of wheat in Germany are warranted. In addition, it is important to note that our results contrast the results from previous studies (Farr and Rossman, 2021) which reported F. venenatum primarily as a soil saprophyte and presumably non-pathogenic fungus in its nature. Our findings thus, also warrant the need to further investigate pathogenic potential of this species and the role it may play on other common rotational crops in Germany.

Arbuscular mycorrhizal fungi modulate the chromium distribution and bioavailability in semi-aquatic habitats

• Arbuscular mycorrhizal fungi (AMF) improve the wetland plant tolerance to Cr stress. • Cr transformation from roots to shoots decrease in AMF inoculated semi-aquatic habitats. • AMF can reduce Cr bioavailability and Cr (VI) concentrations in substrates. • AMF beneficial for the reduction of Cr content in water under fluctuating water depth. Arbuscular mycorrhizal fungi (AMF) can improve plant tolerance to heavy metal stress in the terrestrial system. However, the detoxification ability of AMF to heavy metals in aquatic systems remains poorly understood. This study investigated the effects of AMF on chromium (Cr) distribution and bioavailability in semi-aquatic habitats under different water depths. Results showed that AMF increased the Cr accumulation in the roots of Iris pseudacorus by 10.0–20.0%. Conversely, Cr concentrations in the shoots of AMF inoculated I. pseudacorus were 21.7–68.4% lower than those in the non-inoculated controls. Besides, Cr concentrations and mass contents in water were decreased by 34.1–35.3% and 15.4–23.3% under the low Cr stress (5 mg kg −1 ) with AMF inoculation compare to the non-inoculated treatments, respectively. Moreover, Cr (VI) concentrations in substrates under the Cr contents of 5 mg kg −1 were 27.0–44.0% lower than those under the non-inoculated controls. Meanwhile, AMF reduced the Cr bioavailability in substrates under the fluctuating water depth (water depth of 6–3 cm), with an acid-soluble state of Cr decreased by 4.1–5.6%. Furthermore, AMF also enhanced the biomass, nutrient contents (TC, TN, Ca, Mg, Fe, and Mn) in the I. pseudacorus under the fluctuating water depth. Therefore, it provides a possibility that AMF can be used to remove heavy metals from polluted wastewater by semi-aquatic habitats under fluctuating water depth (e.g. tidal flow constructed wetlands).

Evaluation of the GENE-UP® EHEC Detection Method for the Detection of Enterohemorrhagic E. coli in Select Foods: Collaborative Study: First Action Method 2020.06.

The GENE-UP® EHEC assay (Performance Tested MethodSM 121806) is a real-time PCR molecular detection method that utilizes fluorescence resonance energy transfer proprietary hybridization probes for the rapid detection of enterohemorrhagic Escherichia coli (EHEC) in select foods. The purpose of this validation was to evaluate the method's interlaboratory performance and submit the results to AOAC INTERNATIONAL for adoption as a First Action Official Method of AnalysisSM for the detection of EHEC in select foods. The GENE-UP method was evaluated in a multi-laboratory study as part of the MicroVal VALIDATION certification process using unpaired test portions for one food matrix, raw ground beef (85% lean). Collaborators evaluated the candidate method using either an automated or manual lysis procedure. The candidate method was compared to the ISO/TS 13136:2012 method. Data from 17 participants from 15 laboratories throughout the European Union were evaluated. Three levels of contamination were evaluated: a non-inoculated control level (0 CFU/test portion), a low contamination level (∼1 CFU/test portion), and a high contamination level (∼10 CFU/test portion). Data from the study were analyzed according to the probability of detection (POD) statistical model. The difference in laboratory probability of detection (dLPODC) values with 95% confidence interval between the candidate and reference method results were -0.01 (-0.04, 0.02), 0.23 (0.07, 0.39), and 0.06 (0.01, 0.12) for the non-inoculated, low, and high contamination levels, respectively. For the candidate method, values obtained for repeatability and reproducibility were similar to the reference method and indicated minimal variation between samples or between laboratories. No discrepant results (false positive or false negative) were observed for each contamination. A statistical difference was calculated between the candidate and reference method at the low and high inoculation levels, with the candidate method detecting a higher number of positive samples indicating a higher sensitivity than the reference method. No differences in the recovery of the target analyte were observed between the manual and automated lysis procedures. The GENE-UP EHEC Detection Method provides end users a rapid, easy-to-use workflow for the detection of EHEC in food matrixes.

Evaluation of day of hatch exposure to various Enterobacteriaceae on inducing gastrointestinal inflammation in chicks through two weeks of age

Inappropriate microbial colonization can induce gastrointestinal (GI) inflammation may predispose poultry to opportunistic infections and reduce growth performance. Four independent experiments were completed to test ability of select Enterobacteriaceae isolates to induce GI inflammation. Experiments 1 and 2 included a non-inoculated control (NC), and a low (L), medium (M), or high (H) day of hatch (DOH) oral inoculation level. In experiment 1, birds in L1, M1, and H1 received 102 to 104 CFU of a mixed dose of 2 species of Citrobacter and Salmonella Enteritidis LB (SE). In experiment 2, birds in L2, M2, and H2 received 103 to 105 CFU of E. coli LG (LG) and included NC. Body weight was recorded on d 0, 7, and 14, with blood collected for chicken serum alpha-1-acid glycoprotein (A1GP) measurements on d14. Neither experiment resulted in differences in BWG, however, A1GP was increased (P < 0.05) on d 14 when DOH inoculation dose 103 CFU/chick was used compared to NC. This observed increase in A1GP resulted in selection of 103 CFU/chick for DOH inoculation in experiments 3 and 4. Experiment 3 consisted of NC, E. coli Huff (Huff), and SE. On d 0, 7 and 15, BW was measured, with blood collected on d 15 for A1GP. Both d 15 A1GP and BWG from d 7 to 15 were reduced in inoculated chicks, Huff and SE, in experiment 3 (P < 0.05). Experiment 4 evaluated NC and LG with BW measured on d 0, 2, 7 and 14. Yolk sacs were evaluated for retention and bacterial enumeration, and blood for serum A1GP were collected on d 2 and 14. Experiment 4 resulted in no differences in yolk sac parameters or A1GP, whereas there was an increase in BWG for LG from d 0 to 14 (P < 0.05). When evaluated over time, serum A1GP increased between d 2 and d 14 by nearly 46% in LG, compared to negligible changes in NC (P = 0.111). Mild GI inflammation induced by early Enterobacteriaceae exposure may not drastically impact growth or inflammation parameters but may increase susceptibility to opportunistic infection necessitating further study of this model.

Effects of induced water deficit and biofertilization on growth dynamics and bulb yield of onion (Allium cepaL.) in a neotropical semi-arid environment

In a scenario of world population increase and climate change, an efficient use of water is key for agricultural production. Onion is one of the most profitable crops and can adapt to particular conditions of water stress. The objective of this research was to determine growing degree-days and accumulated radiation under non-stress conditions and yield of an F1 2000 hybrid of onion (Allium cepa L.) under water deficit (WD) and biofertilization in a semi-arid environment. An established nutrient requirement of 247 kg N, 240 kg P2O5(105 kg P), 240 kg K2O (199 kg K), and two irrigation factors were applied: normal irrigation with a daily and WD with a 3 d interval irrigation frequencies. The effect of biofertilization was evaluated through the inoculation of a microbial consortium (MC) in combination with four NPK fertilizer treatments. The crop accumulated 1334 degree-days and 1188 MJ m−2·d−1at the time of harvest at 71 d after transplanting. The yield was 36 t·ha−1, similar under both irrigation conditions; and the WD treatment resulted in a 35% water savings, a 47% and 65% increase in water use efficiency and modulus of elasticity, respectively. The MC resulted in a 50% NPK savings under non-limiting water conditions and produced a similar yield compared with the 100% NPK non-inoculated control. The lower irrigation frequency together with the 100% NPK fertilization dose without the MC and the use of the microorganisms and the 50% NPK treatment without water stress are recommended as agrosustainable practices for onion production.

Enhancement of soil health, germination and crop productivity in Andrographis paniculata (Burm.f.) Nees, an important medicinal crop by using a composite bio inoculant

The untapped microbial communities in medicinal plants can have a genetically varied population with multi-functional plant growth promoting characters. An attempt was made to investigate the effect of bioinoculant, Cohnella sp., Chryseobacterium taklimakanense, Lysobacter soli and Paenibacillus glycanilyticus isolated from the medicinal plant (Hemidesmus indicus) rhizosphere on the growth parameters of Kalmegh (Andrographis paniculata) without chemical fertilizers. A vermicompost carrier based single, dual and multiple bio-inoculant formulation was developed and tested for the survival of individual strains, showed a maximum population of 1 × 106 cells g−1 after 90 days of storage at 28 ± 2 °C invariably in all formulations. The inoculums’ efficiency on Kalmegh under greenhouse conditions resulted in boosted growth with the maximum plant height (95.8 cm) in Cohnella sp. application, followed by consortium of all strains recorded 91.5 cm. Flower initiation occurred sooner in plants inoculated with bacterial consortium of all as well in Cohnella sp. alone, consecutively resulted in highest Andrographolide content of 3.06% and 3.50%, respectively. Maximum fresh weight herbage yield of 39.5% and 27.5% (dry weight) was recorded in plants treated with bacterial consortium (Cohnella sp, C. taklimakanense, L. soli and P. glycanilyticus) over non-inoculated control as well the available nitrogen, phosphorus and potassium content. Germination of seed experiment revealed the synergism of bacterial isolates in consortium for promoting plant growth. Further, the quanta of inoculums is reduced by 25 percent in composite inoculation, in turn reduces the fertilizers expenditure and persist until the harvesting stage of the crop with the need to apply once.