Non-inoculated Control Treatments Research Articles

Occurrence of root rot caused by Fusarium fujikuroi and Fusarium proliferatum on peanut in China.

Root rot of peanut, caused by Fusarium spp., is a devastating disease in most peanut cultivation regions. In this reported outbreak, Fusarium root rot of peanut has been observed in Henan province, China in July 2021. About 20% of peanuts in a field (0.66 ha) were affected. Early symptoms comprised the wilt of the lower leaves, and the darkening of the vascular tissue of roots, which turned brown. Progressively, the whole plant wilted, the roots rotted, and the plant ultimately died. Pathogenic species were isolated from plants showing symptoms of root rot in the field. A total of 206 Fusarium isolates were generated, and 16 isolates were preliminarily identified as Fusarium fujikuroi based on morphological characteristics. Isolates were obtained and grown on PDA plates. Isolates developed floccose white aerial mycelia with reddish-pink coloration in the medium in 2 weeks on the benchtop. Macroconidia were 3-5 septate, measuring 27.5 to 48.8 × 2.6 to 3.8 μm (avg. 36.7 × 3.6 μm, n=50). Microconidia were abundant in chains, mainly asepatate, oval to kidney-shaped, 4.0 to 11.6 × 2.5 to 4.1 μm (avg. 5.8 ×3.1 μm, n=50). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcribed spacer (ITS) region using ITS1/ITS4 primers (White et al.1990) (Genbank assessions MZ831304 to MZ831308), the partial calmodulin gene (CAM, primer CL1/CL2A, O'Donnell.)(Genbank assession MZ856333 to MZ856337) and the partial translation elongation factor (EF-1α) using primer EF1/EF2 (Geiser et al.)(Genbank assession MZ856338 to MZ8564342). FUSARIUM-ID analysis showed 98.18% to 100% similarity with sequences of the F. fujikuroi species complex. The phylogenetic analysis was conducted using a neighbor-joining algorithm based on the ITS, CAM, and EF-1α gene sequences. The isolates were clustered with F. fujikuroi clade (Supplementary Fig.1). Koch's postulates were conducted using a sand-cornmeal-inoculum-layer method (Bilgi et al.). Briefly, 400 ml plastic boxes were filled with 15g of sterilized premium-grade coarse vermiculite, followed by a 15 g of inoculum prepared as sand-cornmeal mixture inoculum. The inoculum for each F. fujikuroi isolates was prepared by infesting a pre-sterilized sand-cornmeal mixture with three 5 mm plugs of cultures. Three F. fujikuroi isolates and PDA plugs were inoculated to serve as positive control and non-inoculated control treatments, respectively. The completed colonization of the sand-cornmeal mixture was finished by incubating at 25 ℃ for 7-10 days. Eight pre-germinated seeds of cv. Luhua No.1 was then covered with another 8 g of vermiculite. Peanuts were grown at 25 °C with 85% relative humidity under a light/dark cycle of 14h/10h. After 14 days of incubation, the inoculated plants showed typical symptoms of root rot similar to those in the field: pre-emergence damping-off, reddish-brown lesions on the tap, and lateral roots. F. fujikuroi was successfully re-isolated from inoculated plants but not from the controls and identified as described above. F. fujikuroi was reported to cause bakanae disease of rice (Amatulli et. al.), and root rot of soybeans (Zhao et. al.). To the best of our knowledge, this is the first record of F. fujikuroi causing root rot of peanut in China.

Symptomless turnip yellows virus infection causes grain yield loss in lentil and field pea: A three-year field study in south-eastern Australia.

Turnip yellows virus (TuYV) is a damaging virus that is persistently transmitted by aphids and infects a wide range of grain hosts including lentil (Lens culinaris Medik), field pea (Pisum sativum L.) and canola (Brassica napus L., oilseed rape). Although information is available about the effects of TuYV infection on grain yield in canola, data about its impact on yield in pulses is lacking. In this study, field experiments quantifying the effects of TuYV infection on the grain yield of lentil and field pea were conducted over three consecutive years (2018-2020) with varying weather conditions. Plants artificially inoculated with TuYV using viruliferous green peach aphid (Myzus persicae, Sulzer) were grown under typical field conditions in south-eastern Australia. At maturity, grain yield, along with associated grain and plant growth parameters, were measured. Compared to the non-inoculated control treatment, early TuYV infection reduced grain yield by up to 36% in lentil and 45% in field pea, while late TuYV infection had no significant impact on yield. Despite a high incidence of TuYV infection and significant yield losses recorded in inoculated plots, no obvious symptoms of virus infection were observed in the inoculated plots in any of the six experiments; this lack of visible symptoms in lentil and field pea has significant implications for crop health assessments, demonstrating the importance of testing for virus instead of relying solely on the presence of visual symptoms, and may also be leading to an underestimation of the importance of TuYV in pulses in Australia.

Synergism between feremycorrhizal symbiosis and free-living diazotrophs leads to improved growth and nutrition of wheat under nitrogen deficiency conditions

A controlled-environment study was conducted to explore possible synergistic interactions between the feremycorrhizal (FM) fungus Austroboletus occidentalis and soil free-living N2-fixing bacteria (diazotrophs). Wheat (Triticum aestivum) plants were grown under N deficiency conditions in a field soil without adding microbial inoculum (control: only containing soil indigenous microbes), or inoculated with a consortium containing four free-living diazotroph isolates (diazotrophs treatment), A. occidentalis inoculum (FM treatment), or both diazotrophs and A. occidentalis inoculums (dual treatment). After 7 weeks of growth, significantly greater shoot biomass was observed in plants inoculated with diazotrophs (by 25%), A. occidentalis (by 101%), and combined inoculums (by 106%), compared to the non-inoculated control treatment. All inoculated plants also had higher shoot nutrient contents (including N, P, K, Mg, Zn, Cu, and Mn) than the control treatment. Compared to the control and diazotrophs treatments, significantly greater shoot N content was observed in the FM treatment (i.e., synergism between the FM fungus and soil indigenous diazotrophs). Dually inoculated plants had the highest content of nutrients in shoots (e.g., N, P, K, S, Mg, Zn, Cu, and Mn) and soil total N (13–24% higher than the other treatments), i.e., synergism between the FM fungus and added diazotrophs. Root colonization by soil indigenous arbuscular mycorrhizal fungi declined in all inoculated plants compared to control. Non-metric multidimensional scaling (NMDS) analysis of the bacterial 16S rRNA gene amplicons revealed that the FM fungus modified the soil microbiome. Our in vitro study indicated that A. occidentalis could not grow on substrates containing lignocellulosic materials or sucrose, but grew on media supplemented with hexoses such as glucose and fructose, indicating that the FM fungus has limited saprotrophic capacity similar to ectomycorrhizal fungi. The results revealed synergistic interactions between A. occidentalis and soil free-living diazotrophs, indicating a potential to boost microbial N2 fixation for non-legume crops.

Inoculation of Arbuscular Mycorrhizal Fungi Improves Growth and Photosynthesis of Ilex paraguariensis (St. Hil) Seedlings

Yerba mate (Ilex paraguariensis St. Hill.) is an important woody tree in South America, however the production of quality seedlings is a major problem. Microorganisms that promote plant growth have shown to be efficient biotechnologies in the production of seedlings. The arbuscular mycorrhizal fungi (AMF), it establishes symbiosis with several plant species and increases the accumulation of biomass and absorption of poorly mobile nutrients such as phosphorus. Here, we test the effects of two AMF species on growth, root architecture, phosphorus accumulation and gas exchange of yerba mate seedlings cultivated under different phosphorus levels. We used seedlings in a non-sterile soil, inoculated with AMFs Rhizophagus clarus SCT720A and Acaulospora colombiana SCT115A, and non-inoculated (control) treatment under five levels of phosphorus (0; 25; 50; 100 and 200% recommendation). After 90 days of AMF inoculation, plant dry biomass, root architecture and mycorrhizal colonization were determined and after 180 days, the same parameters plus photosynthetic rate, transpiration rate, stomatal conductance and P content were evaluated. AMF inoculation increased shoot and root dry biomass, total root length, root volume. Plants inoculated with AMF showed higher photosynthesis rate. Phosphorus content and mycorrhizal colonization were increased almost three times when inoculated with AMF. Our findings highlight the importance of AMF inoculation for Ilex paraguariensis seedlings production, reducing the time needed in nurseries to enhance tree performance.

Effect of bacterial endophytes inoculation on morphological and physiological traits of sorghum (Sorghum bicolor (L.) Moench) under drought

Globally, drought stress causes the negative impact on agriculture production. However, microbial inoculation is the suitable cost-effective technology to attenuate the drought stress. The aim of this experiment was conducted to evaluate the impact of microbial inoculation on morpho-physiological characters of sorghum under moisture stress condition. A pot experiment with sorghum was conducted to access the role of previously identified bacterial endophyte strains. For comparison, two non-inoculated control treatments (T1: absolute control and T2: drought control) were maintained to compare the drought and inoculation effect. Drought was imposed at flowering stage and soil moisture was measure at frequent days. When the soil moisture attains close to zero, plants were rehydrated. During drought and rehydration period, plant morphological and physiological characters were evaluated. Drought stress drastically reduces the plant characters in control (T2-uninoculated) plant. Whereas, in the presence of bacterial inoculation considerably reduces the drought effect also Bacillus sp. inoculation treatment promoted better recovery during their rehydration process than absolute control. Overall, Bacillus sp. inoculation was the most promising bacterium for possible field trials.

Alleviating the deleterious effects of soil salinity and alkalinity on faba bean (Vicia faba L.) production using Rhizobium/Agrobacterium inoculants

ABSTRACT The effectiveness of Rhizobium inoculation is largely determined by soil properties. In this study, the symbiotic efficiency of 17 Rhizobium/Agrobacterium local strains was screened in greenhouse experiments. Subsequently, the combined effects of selected five rhizobial strains plus a starting N-dose (48 kg N ha−1) on nodulation and yield of faba bean were evaluated in two-year field trials. The field experiments were conducted in multiple locations which represent fertile, alkaline-calcareous, and salt-affected soils. Regardless of soil type, rhizobial inoculation overwhelmingly improved the nodulation of faba beans compared to the non-inoculated control and N-fertilized (48 and 96 kg N ha−1) treatments. In all of the sites, inoculated faba beans, with at least two strains, produced grain yield and grain N yield significantly greater, or comparable to N-fertilized (96 kg N ha−1) treatment. The maximum response of faba bean to inoculation was identified in the salt-affected soil, whereas faba bean inoculated with Rlv strain NGB-FR 126 showed significant increases in the averages of grain yield (50%) and grain N-yield (56%) compared to N-fertilized (96 kg N ha−1) treatment, over the two cropping seasons. Besides enhancing nodulation, application of rhizobial inoculants effectively improved production and plant response in alkaline-calcareous and salt-affected soil conditions.

Biochemical responses of mycorrhizal-inoculated Lamiaceae (Lavender, Rosemary and Thyme) plants to drought: a field study

ABSTRACT Mycorrhizal symbiosis may help plants to compensate the water deficit-induced macronutrients, secondary metabolites and osmotic adjustments as the basis of crop production. The present investigation was undertaken to evaluate the physiological responses of the lavender (Lavandula officinalis L.), rosemary (Rosmarinus officinalis L.) and thyme (Thymus vulgaris) host plants to inoculation with mycorrhizal fungi (Funnelliformis mosseae, Rhizophagus irregularis compared with non-inoculated control treatment) in different irrigation levels (irrigation at 75% field capacity, 50% field capacity and rainfed). The 2-year (2015–2016) factorial experiment was conducted based on a randomized complete block design with three replications at Urmia University. The results showed that the stress treatments promoted the generation of osmotic adjustment (total soluble sugars and proline) in non-inoculated plants more than plants inoculated with fungi species. Nutrient (nitrogen, phosphorous and potassium) concentrations and leaf chlorophyll, reduced by water-limiting irrigation, were enhanced with mycorrhizal inoculation. Colonized plants were better adapted and had greater relative water content under stressful conditions due to higher absorption sites for water and nutrients. Also, stress treatments stimulated the phenylalanine ammonia-lyase, DPPH radical-scavenging activity and, the total phenolic and flavonoids content in non-inoculated plants more than that inoculated with two fungi species.

Bacteria from black crusts on stone monuments can precipitate CaCO3 allowing the development of a new bio-consolidation protocol for ornamental stone

The microbial precipitation of CaCO3 is an emerging strategy for the consolidation of decayed limestone. Strains of Bacillus sp. and Micrococcus sp. isolated from black crusts were selected by polycyclic aromatic hydrocarbon enrichment culture and tested for their ability to precipitate CaCO3 by induced mineralization. Among the isolates, Bacillus sp. A2, B1, B5 and Micrococcus sp. A10 produced the largest quantities of biogenic calcite. Stone bio-consolidation was then tested using Carbogel as a carrier. The most efficient bio-cementation on limestone slabs was achieved by Bacillus sp. A2 and B1 and Micrococcus sp. A10. Bio-consolidation treatments were also applied to fragmented stones from the Church of Santa Maria dei Miracoli by inoculating the three bacterial strains individually or as a mixture. Bacillus sp. A2, Micrococcus sp. A10 and the mixture achieved the most efficient consolidation. Our data showed that Carbogel provides an appropriate substitute for natural biofilm formation in bio-consolidation protocols. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed calcified bacterial cells filling the pores of stone specimens, increasing the calcium content by 100% more than a non-inoculated control treatment under the same conditions. Our new bio-consolidation protocol therefore provides an effective strategy to prevent the further deterioration of stone monuments.

Inoculation of plant growth-promoting bacteria attenuates the negative effects of drought on sorghum.

Microbial inoculants are suitable cost-effective technology to help plants endure drought. For the development of commercial inoculants, screening of efficient plant growth-promoting bacteria (PGPB) is a crucial step. The aim of this study was to evaluate the performance of PGPB to modulate drought resistance in Sorghum bicolor. A pot experiment with sorghum was conducted to access the role of previously selected PGPB strains. In addition, two non-inoculated control treatments (with and without urea fertilization) were also evaluated. For comparison, a fully irrigated treatment (FIT) was also assessed. All plants were fully irrigated for 47days when the water supply was completely suspended for the drought-stressed treatments. When the soil moisture was close to zero, the irrigation was resumed. During dehydration and rehydration process, the leaf gas exchange (LGE) was evaluated. The parameters of plant growth and nitrogen nutrition were assessed 8 days after reirrigation. Comparing to the FIT, all treatments reduced the LGE rates, but in the presence of Bacillus sp. ESA 402 photosynthesis rate was less reduced. Some inoculation treatments promoted better recovery of photosynthesis, comparable to the FIT, 6 days after rehydration. The plant growth and nitrogen nutrition were negatively affected by the drought, but the inoculation of different bacteria reduced some negative effects. The nitrogen accumulation in the shoots was increased by all strains, suggesting their diazotrophic ability even under drought. Overall, the inoculation of Bacillus sp. ESA 402 was the best bacterium with potential for future field trials.

Endophytic nitrogen fixation - a possible 'hidden' source of nitrogen for lodgepole pine trees growing at unreclaimed gravel mining sites.

Lodgepole pine (Pinus contorta var. latifolia) trees have been thriving on unreclaimed gravel mining sites in British Columbia, Canada, with tissue nitrogen-content and growth-rate unaffected by extremely low soil nitrogen-levels. This indicates that pine trees could be accessing a hidden nitrogen source to fulfill their nitrogen requirements - possibly via endophytic nitrogen-fixation. Endophytic bacteria originally isolated from native pine trees growing at gravel sites were selected (n = 14) for in vitro nitrogen-fixation assays and a year long greenhouse study to test the overall hypothesis that naturally occurring endophytic nitrogen-fixing bacteria sustain pine tree growth under nitrogen-limited conditions. Each of the 14 bacteria colonized the internal tissues of pine trees in the greenhouse study and fixed significant amounts of nitrogen from atmosphere (23%-53%) after one year as estimated through 15N isotope dilution assay. Bacterial inoculation also significantly enhanced the length (31%-64%) and biomass (100%-311%) of pine seedlings as compared to the non-inoculated control treatment. In addition, presence of the nifH gene was confirmed in all 14 bacteria. Our results support the possibility that pine trees associate with nitrogen-fixing bacteria, capable of endophytic colonization, to survive at unreclaimed gravel mining pits and this association could potentially be utilized for effective reclamation of highly disturbed sites in a sustainable manner.

First Report of Botrytis pelargonii Causing Postharvest Gray Mold on Fresh Ginseng Roots in China

Chinese ginseng (Panax ginseng) is a perennial Chinese herb of the Araliaceae family. It is found in the three northeast provinces of China. As a valuable restorative tonic, it is widely used in traditional Chinese medicine (Radad et al. 2006). In November 2015 and 2016, gray mold was observed on 10 to 35% of fresh ginseng roots in two storage facilities under complete darkness at 4°C in Ji An and Fu Song City, Jilin Province. Initial symptoms included light brown, water-soaked lesions on ginseng roots. Within 30 days, the diseased areas enlarged, with dense, abundant gray mycelia forming on lesions. To isolate the pathogen, small pieces of diseased roots were surface disinfested in 3% NaClO for 2 min, rinsed twice in distilled water, plated on potato dextrose agar (PDA), and incubated at 22°C for 5 days. Sixteen isolates were recovered by single-spore isolation. Colony morphology was almost identical among all isolates. Representative isolate PG-S015 was grown on PDA under constant fluorescent light at 22°C. After 5 days, abundant mycelia with branched conidiophores were produced. Conidia were single, aseptate, pale brown, and globose to subglobose. Conidial size ranged from 8.2 to 17.3 μm in length and from 5.8 to 12.2 μm in width, averaging 13.6 ± 0.6 × 8.7 ± 0.3 μm (n = 50). The conidia were slightly longer than B. cinerea (Zhang et al. 2006). After 9 days, black, scattered, irregular sclerotia were produced. Sclerotial size measured 1.0 to 3.0 × 1.3 to 6.0 mm with the average of 2.1 × 3.8 mm (n = 50). DNA was extracted from all isolates. All 16 isolates were sequenced for the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), DNA-dependent RNA polymerase subunit II (RPB2), and heat-shock protein 60 (HSP60) genetic markers. Their ITS sequences (KY810515 to KY810530) were identical to those of B. pelargonii (AJ716290) and B. cinerea (KY319171). The G3PDH (KY827182 to KY827197) and HSP60 (KY827198 to KY827213) sequences were 137 and 42 bp shorter than those of B. cinerea (GenBank KY275256 and KX229753) and identical to those of B. pelargonii (AJ704990 and AJ716046). The RPB2 sequences (KY827214 to KY827229) were also 99% identical to that of B. pelargonii (AJ745662). The fungus was identified as B. pelargonii based on its morphological and molecular characteristics (Staats et al. 2005; Zhang et al. 2006). Pathogenicity tests were performed by pipetting a 100 μl of conidial suspension (1 × 10⁵ conidia/ml) of each selected isolate PG-S015 and PG-S-FS-017 onto a 5-year-old fresh ginseng root, three replicates per isolate. Sterilized water was used as the noninoculated control treatment. The experiment was carried out three times. All inoculated roots were maintained at 4°C and 90% relative humidity, similar to the environmental conditions in which the disease was observed. Within 30 days, all inoculated ginseng roots developed similar symptoms to those of the naturally occurring disease described above, whereas all noninoculated roots remained healthy. Isolates with identical morphological and molecular characteristics were recovered from the diseased roots. Prior to this report, B. pelargonii was exclusively recovered from Pelargonium spp. (Strider 1985). This is the first report of B. pelargonii causing postharvest gray mold on stored fresh ginseng roots. Proper management should be taken to reduce the losses from this disease to the quality and marketability of ginseng as a highly valuable traditional Chinese medicine.

Arbuscular mycorrhizal fungi and Urochloa brizantha: symbiosis and spore multiplication

ABSTRACT Arbuscular mycorrhizal fungi (AMF) may exhibit distinct behaviors when associated with the same species of host plant, being necessary to understand their ecology, in order to optimize their management and maintenance in germplasm bank. This study aimed to evaluate different AMF associated with Urochloa brizantha (A. Rich.) Stapf by analyzing the symbiosis establishment time, spore multiplication and production of glomalin-related soil protein. The experiment was carried out in a completely randomized design, in a 6 x 5 arrangement (five AMF species, non-inoculated control treatment and five evaluation times). The following aspects were analyzed: plant growth, spore multiplication, mycorrhizal colonization and glomalin production. The highest number of spores occurred for Acaulospora longula and A. colombiana, exhibiting the highest mycorrhizal colonization at 76 days. The inoculation favored the root growth of U. brizantha at 15 days of cultivation, plant height and root dry matter at 60 days and shoot dry matter at 90 days, especially for the Acaulospora species. The inoculation with A. colombiana, A. longula and Paraglomus occultum resulted in increased glomalin at 120 days. Gigaspora margarita and P. occultum did not reach the maximum colonization and spore multiplication, indicating that a period of time longer than 120 days of cultivation is necessary.

Temperature as a key factor for successful inoculation of soybean with Bradyrhizobium spp. under cool growing conditions in Belgium

AbstractBacterial inoculation of soybean seeds to improve biological nitrogen fixation is a well-known practice to achieve higher seed and protein yield with reduced fertilization. The optimal inoculation strategy in temperate regions is unknown because soybeans are rarely cultivated under colder growing conditions. The aim of the present work was to determine the most suitable inoculation strategy for soybean cultivation in Belgium. Field trials were set up with four Bradyrhizobium inoculants (HiStick, Force 48, Biodoz and Optimize) at two locations over 2 years (2014–2015) and compared with a non-inoculated control treatment. In addition, HiStick was tested at three doses and Optimize at two time periods prior to sowing. Under Belgian conditions, all inoculants were effective in establishing rhizobial symbiosis, resulting in increased yield, protein content, protein yield and thousand-grain weight compared with the non-inoculated control. A single dose of HiStick was sufficient to establish symbiosis. Pre-inoculation with Optimize 2 weeks before sowing gave an intermediate performance for most parameters between the non-inoculated control treatment and inoculation with Optimize 24 h prior to sowing. Among the four products tested, Biodoz seemed the best product for inoculation under cool growing conditions. Based on the atpD gene, the bacterial strain of Biodoz showed complete similarity with Bradyrhizobium diazoefficiens, while strains of other products were identified as Bradyrhizobium japonicum. In vitro growing capacity of the Biodoz strain at 8 °C was higher compared with the other strains. Better cold adaptation of the Biodoz strain might be a possible explanation for the better performance of Biodoz in Belgium.

Acumulación de biomasa en yuca (Manihot esculenta C.) ante la respuesta a la aplicación de fertilizantes NPK y biofertilizantes en condiciones de invernadero en dos tipos de suelo

El cultivo de la yuca en el Caribe colombiano es un sistema productivo con un bajo nivel de integración de tecnologías modernas sustentables que permitan incrementar la rentabilidad del cultivo. La validación de prácticas asociadas a la fertilización conforma una de las alternativas para incluir en las estrategias de manejo integrado del cultivo. El objetivo de este estudio fue evaluar a nivel de casa malla, la respuesta de acumulación de biomasa en plantas de yuca a la fertilización NPK en dos tipos de suelos (arenoso y franco-arcilloso) y en presencia de tres biofertilizantes. El efecto de diferentes inoculantes microbianos comercialmente disponibles, Azobac® (Azotobacter chrococcum), Fosforiz® (Pseudomonas fluorescens) y micorrizas arbusculares (Safer Micorrizas ®), fue evaluado en mezcla con diferentes niveles de fertilización inorgánica (100, 75, 50, 25 y 0%), más los testigos sin inoculación en un arreglo factorial 2×3×5. Los resultados obtenidos indican que el nivel de fertilización NPK en el que se alcanza la mayor acumulación relativa de biomasa ocurre al 25% de la mezcla. Los mayores valores de acumulación de biomasa se alcanzan con la presencia de micorrizas. Azobac® y Fosforiz ® no exhibieron ningún efecto sobre la acumulación de biomasa. Los resultados obtenidos sugieren que los biofertilizantes comerciales basados en micorrizas pueden ser usados para maximizar la respuesta a la fertilización con NPK.

Compatible bacterial mixture, tolerant to desiccation, improves maize plant growth.

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

Genetic Variability and Symbiotic Efficiency of Erythrina velutina Willd. Root Nodule Bacteria from the Semi-Arid Region in Northeastern Brazil

Legume-rhizobia symbiosis is a cross-kingdom association that results in large mounts of nitrogen incorporated in food webs. For the Brazilian semi-arid region, data on genetic variability and symbiotic efficiency of Papilionoidae rhizobial communities are very scarce. The aim of this study was to evaluate the genetic variability and the symbiotic efficiency of eight rhizobial isolates obtained from ?mulungu? (Erythrina velutina Willd.) nodules. For 16S rRNA gene sequencing, the genomic DNA was extracted using a commercial kit, amplified with universal primers, and subjected to sequencing reactions. For the isolate ESA 71, PCR amplifications for nodC and nodA genes were attempted. Rhizobial efficiency was assessed by two greenhouse experiments. The first assay was carried out under gnotobiotic conditions, with sterile sand as a substrate; the second experiment was conducted in a non-sterile soil. For both experiments, the inoculation treatments consisted of a single inoculation of each isolate, in addition to a treatment with Bradyrhizobium elkanii BR 5609 as a reference strain. Furthermore, two non-inoculated control treatments, supplied and not supplied with mineral N, were also evaluated. Bacterial identification indicated that both and rhizobia could be found in ?mulungu? root nodules. Three isolates where classified within the Rhizobium genus, four bacteria belonged to Bradyrhizobium and one isolate clustered with Burkholderia. Positive amplification of an intragenic fragment of the nodA gene using a primer set to ?-rhizobia could be found for ESA 71 (Burkholderia). All bacterial isolates were effective in colonizing ?mulungu? roots. In the first experiment, all inoculated treatments and N fertilization increased the N concentration in ?mulungu? shoot tissues. For total N in the shoots, the isolates ESA 70, ESA 72, and ESA 75 stood out. In the non-sterile substrate experiment, the isolates ESA 70, ESA 71, ESA 72, and ESA 75, together with the reference strains, induced increases in the shoot N concentration and total accumulation compared to the absolute control. The results indicate that ?mulungu? is able to establish associations with efficient ? and ?-rhizobia in Brazilian semi-arid soils.

English

Amburana cearensis (Allemao) A.C. Smith is a widely used legume by the population due to its medicinal properties. This species establish symbiosis with the arbuscular mycorrhizal fungi (AMF) that can increase the production of secondary metabolites, a fact which has not been clarified for this plant. Therefore, the aim of this study was to examine the contribution of the AMF in the production increase of foliar bioactive compounds in A. cearensis seedlings. The experiment which under-goes protected roofing was carried out using four inoculation treatments: non-inoculated control treatment, inoculated with Gigaspora albida, inoculated with Claroideoglomus etunicatum and inoculated with Acaulospora longula. After 160 days, the following was examined: dry matter of the aerial part, chlorophylls a, b and total, soluble carbohydrates, total proteins, total phenols, total flavonoids and total tannins. A. cearensis seedlings inoculate with C. etunicatum accumulated more dry matter of the aerial part (78.38%), total chlorophylls (24.28%) and chlorophylls b (53.63%), total phenols (47.82%), total flavonoids (32.28%) and total tannins (61.58%) in relation to the control treatment. Mycorrhizal technology using the C. etunicatum fungus is an alternative to increase the levels of foliar bioactive compounds in A. cearensis seedlings.   Key words: Caatinga, phenolic compounds, arbuscular mycorrhizal fungi (AMF).

First Report of Pythium sulcatum Causing Cavity Spot in Processing Carrot Crops in the Columbia Basin of Washington State.

In October 2012, symptoms of cavity spot (1) were observed on roots of two 50 ha, Red Core Chantenay processing carrot (Daucus carota L. subsp. sativus (Hoffm.)) crops in the Columbia Basin of central Washington. Symptoms consisted of sunken, elliptical lesions (3 to 15 mm long) on the root surface. Approximately 6% of the roots in each crop were affected, which was sufficient to present sorting problems for the processor. Symptomatic roots were washed thoroughly in tap water, and then small sections of tissue from the lesion margins were removed aseptically and plated onto water agar (WA) without surface-sterilization. Isolates with morphological characteristics typical of Pythium sulcatum Pratt & Mitchell (2) were obtained consistently from the symptomatic tissue. The genus and species identity of seven isolates was confirmed by sequence analysis of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA (rDNA) using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B with the PCR protocol described by Schroeder et al. (3). The ITS consensus sequences of the seven isolates (Accession Nos. KF509939 to KF509945) were 98 to 99% homologous to ITS sequences of P. sulcatum in GenBank. Pathogenicity of all seven isolates was confirmed by inoculating mature carrot roots of cv. Bolero. Each root was washed with tap water, sprayed to runoff with 70% isopropanol, and dried in a laminar flow hood on sterilized paper toweling. The roots were then placed in plastic bins lined with paper toweling moistened with sterilized, deionized water. Each root was inoculated by placing two 5 mm-diameter agar plugs, taken from the edge of an actively growing WA culture of the appropriate isolate, on the root surface approximately 3 cm apart. Non-colonized agar plugs were used for a non-inoculated control treatment. Four replicate roots were inoculated for each isolate and the control treatment. After inoculation, the roots were misted with sterilized, deionized water, a lid was placed on each bin, and the roots were incubated in the dark at 22°C. Roots were misted daily to maintain high relative humidity. Dark, sunken lesions were first observed 3 days post-inoculation on roots inoculated with the P. sulcatum isolates, and all inoculated roots displayed cavity spot lesions by 7 days. No symptoms were observed on the non-inoculated control roots. Colonies with morphology typical of P. sulcatum were re-isolated from the symptomatic tissue of roots inoculated with the P. sulcatum isolates, and the species identity of the re-isolates was confirmed by ITS rDNA sequence analysis, as described above. Although P. sulcatum is one of several Pythium species that can cause cavity spot of carrot (1), to our knowledge, this is the first report of P. sulcatum causing cavity spot in Washington State, which has the largest acreage of processing carrot crops in the United States (4).

First Report of Pythium recalcitrans Causing Cavity Spot of Carrot in Michigan.

During a survey of carrot (Daucus carota L.) cavity spot in Michigan in September 2010, carrot roots with typical cavity spot symptoms were collected from production fields in Fremont Co. The lesions were excised from infected roots, surface-disinfested with 0.62% NaClO for 3 min, rinsed in sterilized, distilled water three times, cut into 0.5 cm long pieces, and then plated on water agar (WA) amended with carbendazim (10 μg/ml), ampicillin (50 μg/ml), rifampicin (50 μg/ml), and pentachloronitrobenzene (10 μg/ml) (cumulatively referred to as CARP). Plates were incubated at 22 ± 1°C in the dark for 3 days. Pure cultures of the isolates were obtained by transferring a single hyphal tip of each colony to potato dextrose agar (PDA) amended with CARP. Among the 33 isolates obtained, M2-05 was identified as a Pythium sp. that differed from the known cavity spot pathogens of carrot. The isolate has spherical hyphal swellings but no other distinguishing morphological characteristics. M2-05 was further classified by analyzing the partial sequences of four genes: the internal transcribed spacer (ITS) region of ribosomal DNA, beta-tubulin (β-tub), cytochrome c oxidase subunit 2 (cox 2), and NADH dehydrogenase subunit 1 (nadh 1) (1,3). A BLAST search of these sequences for M2-05 was conducted using the nucleotide database of GenBank, resulting in 100% similarity to all four sequenced genes of P. recalcitrans (2). The DNA sequences of M2-05 were deposited in GenBank (JQ734349, JQ734229, JQ734289, and JQ734409 for ITS, β-tub, cox 2, and nadh 1, respectively). Koch's postulates were conducted by inoculating mature carrot roots (cv. Nantindo) with mycelial plugs (4 mm in diameter) cut from the margin of actively growing colonies of M2-05 on PDA plates. Two mycelial plugs were placed on each carrot root at 3-cm intervals, with the mycelial side facing the root; and two non-colonized agar plugs were placed similarly for the non-inoculated control treatment. In comparison, carrot roots also were inoculated with an isolate of each of P. sulcatum and P. violae using the same method. There were four replicate carrot roots inoculated for each isolate and each of the control treatments. The inoculated roots were placed on a plastic grid (7 mm in height) in a closed plastic container, with moist paper towels underneath the grids. The container was incubated in the dark at 22 ± 1°C, and the roots were sprayed gently daily with sterilized, distilled water to maintain high humidity. Brown lesions were observed on all inoculated carrot roots 5 days after inoculation. The lesions measured 0.68 ± 0.48, 1.20 ± 0.71, and 0.56 ± 0.31 mm2 averaged over all eight lesions for the isolates of P. recalcitrans, P. sulcatum, and P. violae, respectively. Symptomatic tissues from the inoculated roots were excised and incubated on WA-CARP plates, and the culture from each lesion confirmed as the isolates inoculated using the same molecular methods described above. The carrot tissue under the control agar plugs remained symptomless, and no Pythium was recovered from the control roots. P. recalcitrans was described in 2008 as infecting roots of Beta vulgaris and Vitis vinifera (2). To our knowledge, this is the first published report of P. recalcitrans naturally infecting carrot roots, not only in Michigan, but anywhere in the world.

First Report of Daylily Leaf Streak Caused by Kabatiella microsticta in China.

Daylily (Hemerocallis spp.) is an herbaceous, perennial plant, cultivated for its flowers. Daylily is sold in Asian markets as fresh or dried flowers (the flowers of some species, e.g., Hemerocallis citrina, are edible) or as the corm, which is used for medicinal purposes. In June 2011, daylily leaf streak was found in a nursery of Jilin Agricultural University, Jilin Province, China. Symptoms included water-soaked, irregular spots along the leaf midvein that turned orange to reddish brown and eventually enlarged to coalesce into extensive, necrotic streaks along the length of the leaf, as previously reported (2). Heavily infected leaves often withered and died. Four isolates were recovered from necrotic tissue of leaf spots and cultured on potato dextrose agar (PDA) at 25°C. All colonies were initially cream to peach colored and appeared slimy. With the maturation of the culture, the colonies became dark brown to black with sparse aerial hyphae. Blastic conidia formed simultaneously on intercalary or terminal, undifferentiated conidiogenous cells, and were scattered in dense sections on culture surface. When the conidia dropped from conidiogenous cell, an indistinct scar or a denticle remained. Conidia were hyaline, one-celled, smooth, ellipsoidal, and variable in size (2.73 to 6.01 × 8.45 to 19.36 μm), and all morphological characteristics were consistent with Kabatiella microsticta Bubak (syn. Aureobasidium microstictum; 2,4). The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified using primers ITS4/ITS5 (1). ITS (534 bp) was identical among all four isolates (GenBank Accession No. HE798117) and 100% identical to that of K. microsticta CBS 114.64 (FJ150873). Pathogenicity was confirmed by spraying 20 seedlings of daylily, propagated in tissue-culture medium, with a conidial suspension (106 conidia/ml) of each isolate. A second set of 20 seedlings was sprayed with the same volume of sterile water as the noninoculated control treatment. Plants were grown in the greenhouse at 20 to 25°C and were covered with plastic bags to maintain humidity on the foliage for 72 h. After 5 days, the foliar symptoms described earlier for the field plants appeared on the leaves, whereas the control plants remained healthy. K. microsticta was reisolated from the leaf spots of all 20 inoculated plants. Leaf streak is the most destructive disease of daylily, and was previously reported in Japan and the United States (Illinois, Kentucky, Mississippi, Louisiana, Pennsylvania, Maryland, Virginia, Florida, North Carolina, and Georgia) (3). To our knowledge, this is the first report of the disease caused by K. microsticta in China.