Surface-sterilized Seeds Research Articles

Cultivable Endophytic Bacteria in Seeds of Dongxiang Wild Rice and Their Role in Plant-Growth Promotion

Dongxiang wild rice (Oryza rufipogon Griff.) germplasm is a precious resource for the improvement of agronomic traits in rice. Rice seeds also harbor a diverse endophytic bacterial community, and their interactions with their hosts and each other can influence plant growth and adaptability. Here, we investigated the community composition of cultivable endophytic bacteria obtained from the surface-sterilized seeds of Dongxiang wild rice and screened them for plant growth-promoting traits. Phylogenetic analysis of 16S rRNA gene sequences indicated that the 47 isolates were affiliated with five classes and 13 discrete genera, and Bacillus and Microbacterium predominated. Evaluations of plant growth promoting (PGP) traits showed that 45 endophytic bacteria isolates produced between 3.37 and 90.11 μg mL−1 of Indole-3-acetic acid (IAA), with the highest yield of 90.11 μg mL−1 (Fse28). Further, 37 of the isolates were able to solubilize mineral phosphate, while 28 other isolates had the ability of N2-fixation, 17 isolates possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity with the highest yield of 20.72 μmol mg−1 protein h−1 (Fse35), and 17 isolates were also able to produce siderophores. The two strains Fse28 and Fse35 had multiple PGP traits that significantly improved the agronomic traits (root length, shoot length, dry matter, and chlorophyll content) of cultivated rice seedlings. Our results illustrate the rich diversity of seed endophytic bacteria in Dongxiang wild rice and their potential for developing novel efficient bioinoculants to enhance soil fertility and favor seedling growth.

Alpha-tocopherol reinforce selenium efficiency to ameliorates salt stress in maize plants through carbon metabolism, enhanced photosynthetic pigments and ion uptake

Individually selenium and α-tocopherol showed a pivotal role in combating abiotic stresses in plants. However, there is no report on how selenium behaves in the presence or absence of α-tocopherol under salt stress in crop plants. With this view, the present study was undertaken to dissect the interaction between selenium and α-tocopherol on growth performance, carbon metabolism, and uptake of different ions in maize plants grown under salt stress. This study was conducted with surface-sterilized seeds of maize that were soaked in deionized water (control), selenium (0.5 µM), and/or α-tocopherol (200 ppm) for 12 h before sowing and different salt levels (0, 100, 150, and 200 mM) in the form of NaCl was created in soil. A sample of maize plants from each treatment was collected 40 days after sowing. The results revealed that salinity lowered the growth performance, chlorophyll content, insoluble sugar, carbohydrate, phenolic, flavonoid content, and different ions uptake in concentration dependent manner whereas, soluble sugar, α-amylase activity, and sodium ion increased in comparison to control plants. Moreover, selenium and/or α-tocopherol treated plants without stress significantly amplified the growth performance, chlorophyll and carotenoid content, carbohydrate, phenolic, and flavonoid content, and improved the uptake of different concentrations ions (K+, Ca+2, K+/Na+, Ca+2/Na+, and Mg+2) over the control plants. Interestingly, treatment of combined selenium and α-tocopherol to the salt-stressed plants successfully recover the loss caused by increasing salt levels in maize plants through altered carbon metabolism and ions uptake reflected in improved growth performance and bigger growth leaf area of maize plants under salt stress. Selenium and α-tocopherol in combination also enhance the antioxidative defensive mechanism through the production of phenolics and flavonoids content in maize plants under salt stress. This approach could be an effective measure to lower salt stress and enhance the crop productivity of maize plants.

Effect of Co-Inoculation of Bacillus sp. Strain with Bacterial Endophytes on Plant Growth and Colonization in Tomato Plant (Solanum lycopersicum)

Colonization of a biofertilizer Bacillus sp. OYK strain, which was isolated from a soil, was compared with three rhizospheric and endophytic Bacillus sp. strains to evaluate the colonization potential of the Bacillus sp. strains with a different origin. Surface-sterilized seeds of tomato (Solanum lycopersicum L. cv. Chika) were sown in the sterilized vermiculite, and four Bacillus sp. strains were each inoculated onto the seed zone. After cultivation in a phytotron, plant growth parameters and populations of the inoculants in the root, shoot, and rhizosphere were determined. In addition, effects of co-inoculation and time interval inoculation of Bacillus sp. F-33 with the other endophytes were examined. All Bacillus sp. strains promoted plant growth except for Bacillus sp. RF-37, and populations of the rhizospheric and endophytic Bacillus sp. strains were 1.4–2.8 orders higher in the tomato plant than that of Bacillus sp. OYK. The plant growth promotion by Bacillus sp. F-33 was reduced by co-inoculation with the other endophytic strains: Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6., though the population of Bacillus sp. F-33 maintained or slightly decreased. When Klebsiella sp. Sal 1 was inoculated after Bacillus sp. F-33, the plant growth-promoting effects by Bacillus sp. F-33 were reduced without a reduction of its population, while when Bacillus sp. F-33 was inoculated after Klebsiella sp. Sal 1, the effects were increased in spite of the reduction of its population. Klebsiella sp. Sal 1 colonized dominantly under both conditions. The higher population of rhizospheric and endophytic Bacillus sp. in the plant suggests the importance of the origin of the strains for their colonization. The plant growth promotion and colonization potentials were independently affected by the co-existing microorganisms.

The endophytic microbiota of Citrus limon is transmitted from seed to shoot highlighting differences of bacterial and fungal community structures

Citrus limon (L.) Burm. F. is an important evergreen fruit crop whose rhizosphere and phyllosphere microbiota have been characterized, while seed microbiota is still unknown. Bacterial and fungal endophytes were isolated from C. limon surface-sterilized seeds. The isolated fungi—belonging to Aspergillus, Quambalaria and Bjerkandera genera—and bacteria—belonging to Staphylococcus genus—were characterized for indoleacetic acid production and phosphate solubilization. Next Generation Sequencing based approaches were then used to characterize the endophytic bacterial and fungal microbiota structures of surface-sterilized C. limon seeds and of shoots obtained under aseptic conditions from in vitro growing seedlings regenerated from surface-sterilized seeds. This analysis highlighted that Cutibacterium and Acinetobacter were the most abundant bacterial genera in both seeds and shoots, while Cladosporium and Debaryomyces were the most abundant fungal genera in seeds and shoots, respectively. The localization of bacterial endophytes in seed and shoot tissues was revealed by Fluorescence In Situ Hybridization coupled with Confocal Laser Scanning Microscopy revealing vascular bundle colonization. Thus, these results highlighted for the first time the structures of endophytic microbiota of C. limon seeds and the transmission to shoots, corroborating the idea of a vertical transmission of plant microbiota and suggesting its crucial role in seed germination and plant development.

Growth and Competitive Infection Behaviors of Bradyrhizobium japonicum and Bradyrhizobium elkanii at Different Temperatures

Growth and competitive infection behaviors of two sets of Bradyrhizobium spp. strains were examined at different temperatures to explain strain-specific soybean nodulation under local climate conditions. Each set consisted of three strains—B. japonicum Hh 16-9 (Bj11-1), B. japonicum Hh 16-25 (Bj11-2), and B. elkanii Hk 16-7 (BeL7); and B. japonicum Kh 16-43 (Bj10J-2), B. japonicum Kh 16-64 (Bj10J-4), and B. elkanii Kh 16-7 (BeL7)—which were isolated from the soybean nodules cultivated in Fukagawa and Miyazaki soils, respectively. The growth of each strain was evaluated in Yeast Mannitol (YM) liquid medium at 15, 20, 25, 30, and 35 °C with shaking at 125 rpm for one week while measuring their OD660 daily. In the competitive infection experiment, each set of the strains was inoculated in sterilized vermiculite followed by sowing surface-sterilized soybean seeds, and they were cultivated at 20/18 °C and 30/28 °C in a 16/8 h (day/night) cycle in a phytotron for three weeks, then nodule compositions were determined based on the partial 16S-23R rRNA internal transcribes spacer (ITS) gene sequence of DNA extracted from the nodules. The optimum growth temperatures were at 15–20 °C for all B. japonicum strains, while they were at 25–35 °C for all B. elkanii strains. In the competitive experiment with the Fukagawa strains, Bj11-1 and BeL7 dominated in the nodules at the low and high temperatures, respectively. In the Miyazaki strains, BjS10J-2 and BeL7 dominated at the low and high temperatures, respectively. It can be assumed that temperature of soil affects rhizobia growth in rhizospheres and could be a reason for the different competitive properties of B. japonicum and B. elkanii strains at different temperatures. In addition, competitive infection was suggested between the B. japonicum strains.

Requirements for efficient plantlet regeneration using cotyledonary nodal explants of purple coral tree (Erythrina fusca Lour.)

There are over hundred species in the genus Erythrina which are of practical importance in multiple ways. The requirements for Erythrina plantlet regeneration in vitro were rarely studied. Surface-sterilized seeds of purple coral tree (Erythrina fusca Lour.) exhibited a high germination rate (80%) in vitro. The cotyledonary nodes from the one-month-old purple coral tree seedlings grown in vitro were used as explants. About 70% of the cotyledonary nodal explants exhibited shoot regeneration when the explants were cultured vertically with the cut ends inserted into basal Murashige and Skoog (MS) medium compared to only 10% of those placed lying horizontally on the surface of the medium. Supplementation of the basal MS medium with 1 mg L−1 of BAP, 1 mg L−1 kinetin and 1 mg L−1 TDZ resulted in the greatest shoot elongation and the highest number of shoots developed from the cotyledonary nodal explants. The regenerated shoots were able to root (100%) on basal MS medium while supplementation with 2 mg L−1 of either NAA or IBA was inhibitory to root elongation. This is the first study on plantlet regeneration of purple coral tree which seems to have more complex requirements for shoot regeneration in vitro than those reported for the other three Erythrina species. Since only 30% of the rooted in vitro shoots survived during acclimatization following a previously published protocol for acclimatization of another plant, it is recommended that future studies are to investigate the requirements of higher plantlet survival for the rooted in vitro shoots of purple coral tree.

Aspergillus tubingensis Is a Pre-Emergent Pathogen of Date Palm Seedlings

Many diseases of date palm are known. However, pathogens that might affect seed germination and seedling emergence from soil are poorly studied, perhaps because date palm cultivars are propagated vegetatively. Here, we first determined the effects of date seed fungi on the germination and emergence of 600 seeds overall (i.e., 200 of each of three cultivars: ‘Thoory’, ‘Halawi’, and ‘Barhi’). In each cultivar, 100 seeds were from Saudi Arabia (part of the native range), and 100 were from the southwestern USA (where the date palm was introduced around 1765). Just four fungal genera (i.e., Alternaria, Aspergillus, Chaetomium, and Penicillium) were isolated from the surface-sterilized date seeds. Aspergillus isolates all belonged to Aspergillus sect. Nigri; collectively they were in the highest relative abundance at 39%, and significantly more common in Saudi Arabian seeds than in American seeds. Aspergillus reduced seed germination and also reduced emergence when germinated and non-germinated seeds were planted in potting mix in a greenhouse. In contrast, Penicillium species were more common in American than in Saudi seeds; Penicillium did not affect germination, although it did have a positive effect on seedling emergence. In a second experiment with 17 seeds of the ‘Halawi’ cultivar, fungus-free seeds were either inoculated with isolates of Aspergillus sect. Nigri or not, and then planted. Controls emerged whereas Aspergillus-inoculated seeds did not. Finally, a third experiment was conducted with Aspergillus tubingensis Mosseray, a sect. Nigri member, as sole inoculum of 100 ‘Halawi’ seeds versus 100 uninoculated controls. Aspergillus tubingensis exerted the same pathogenic effects on germinating and emerging seedlings as the isolates identified only to Aspergillus sect. Nigri. Aspergillus tubingensis is thus a previously unreported, seedborne pathogen affecting date palm seedlings. Our findings also suggest that A. tubingensis may be more common in seeds in the host’s native range than in its introduced range.

Bradyrhizobium japonicum, B. elkanii and B. diazoefficiens Interact with Rice (Oryza sativa), Promote Growth and Increase Yield.

Bradyrhizobium is a genus of plant growth-promoting rhizobacteria (PGPR) that have been studied for several decades mainly for the ability to fix diazotrophic nitrogen after having been established endosymbiotically inside root nodules of the legumes of Fabaceae. The aim of this work was to evaluate the capability of Bradyrhizobium to promote the growth of crops belonging to other families, in this case, rice (Oryza sativa), both in laboratory and in field trials. For laboratory test, surface-sterilized rice seeds were soaked with cultures of each strain and planted in pots. Plant length and dry weight were measured after 35days. For the field test, rice seeds of varieties Yeruá La Plata and Gurí INTA were inoculated with the three best strains observed in the laboratory test and planted in plots. After 60days of growth, plant length and dry weight were measured. At harvest time, we measured the dry weight of the aerial part, yield and thousand-grain weight. Inoculation with any of the three species described provoked significant increments compared to the uninoculated control at least in one of the parameters measured, both in the laboratory and in the field tests. Bradyrhizobium japonicum E109 was the strain that promoted rice growth the most in the lab while Bradyrhizobium elkanii SEMIA 587 was the strain that promoted rice growth the most in the field, with increments in yield of approximately 1000kg/ha. Data obtained suggest that the Bradyrhizobium species promoted all rice growth and yield.

Draft Genome Sequences of Six Strains of Lactococcus lactis (Phylum Firmicutes), Spanning the Seeds of Cucumis sativus L. (Cucumber), Cucumis melo L. (Cantaloupe), and Cucurbita pepo var. turbinate (Acorn Squash).

We announce the draft genome sequences of six strains of Lactococcus lactis (EKM101L, EKM102L, EKM201L, EKM203L, EKM501L, and EKM502L). These candidate plant probiotics were isolated from surface-sterilized seeds of Cucumis sativus L. (cucumber), Cucumis melo L. (cantaloupe), and Cucurbita pepo var. turbinate (acorn squash). They display beneficial activities, including biocontrol.

Gracilibacillus oryzae sp. nov., isolated from rice seeds.

A Gram-stain-positive, facultatively anaerobic, endospore-forming bacterium, designated strain TD8T, was isolated from surface-sterilized rice seeds (Oryza sativa L.). Phylogenetic analysis of the 16S rRNA gene indicated that strain TD8T should be placed within the genus Gracilibacillus (95.2-99.0 % sequence similarity); it exhibited highest similarities to Gracilibacillus ureilyticus CGMCC 1.7727T (99.0 %), 'Gracilibacillus xinjiangensis' CGMCC 1.12449T (98.9 %) and Gracilibacillus dipsosauri CGMCC 1.3642T (97.5 %). Chemotaxonomic analysis showed that menaquinone-7 (MK-7) was the major isoprenoid quinone. Diphosphatidylglycerol, phosphatidylglycerol and one unidentified phospholipid were the major cellular polar lipids, and the major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, C16 : 0 and iso-C16 : 0, which supported the allocation of the strain to the genus Gracilibacillus. The digital DNA-DNA hybridization value between strain TD8T and Gracilibacillus ureilyticus CGMCC 1.7727T was lower than 70 % (22.60 %), and the average nucleotide identity score was 79.54±5.09 %, suggesting that strain TD8T represented a novel species in the genus Gracilibacillus. The genomic DNA G+C content was 37.5 %. Based on physiological and biochemical characteristics and genotypic data, strain TD8T represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus oryzae sp. nov. is proposed. The type strain is TD8T (=ACCC 61556T=CICC 24889T=JCM 33537T).

Growth and Nutritional Responses of Cowpea (cv. Soronko) to Short-term Elevated Temperature

Short-term variations in temperature associated with climate change have been noted to affect the physiological processes and metabolite profile of plants, including the nutritional status, ultimately affecting their growth and development. An evaluation of the effects of elevated temperatures on the growth and nutritional quality of cowpea was performed during this experiment. The main objective was to evaluate the effects of short-term elevated temperatures on the nutritional quality of cowpea at different growth stages. Surface-sterilized seeds of cowpea (cv. Soronko) were germinated in pots in the glasshouse. At different growth stages (preflowering, flowering, and postflowering), plants were incubated in growth chambers set at three different temperature regimes (25, 30, and 35 °C) for a period of 7 days. Compared with control (25 °C), exposure to both elevated temperatures (30 and 35 °C) reduced the whole plant fresh weight and dry weight by 30% and 52% and 42% and 29%, respectively, at the preflowering stage, and by 31 and 60% and 47 and 63%, respectively, at the flowering/anthesis stage. However, no significant difference in whole plant biomass was noted between elevated temperatures (35%) and the control temperature at the postflowering stage. Short-term exposure to an elevated temperature (35 °C) increased the shoot crude protein content (5.59 N%) of cowpea compared with control (3.77 N%) and preflowering stage. In contrast, at the flowering stage, an elevated temperature (35 °C) reduced the crude protein content (1.77%) of the shoot compared with control (5.59%). At an elevated temperature (35 °C), the preflowering and flowering stages of cowpea were most affected compared with control. These results suggest that the preflowering and flowering stages of cowpea compared with the postflowering stage are more susceptible to elevated temperatures (30 to 35 °C).

Phylogenetic signal of host plants in the bacterial and fungal root microbiomes of cultivated angiosperms.

Root microbiomes are established through selective recruitment by host plants from pools of potential partners. However, the assembly rules of root microbiomes remain unclear. To elucidate (i) the effects of host plant phylogeny on root microbiome assembly and (ii) which microbial groups affect differences in root microbiome assemblies, the structures of bacterial and fungal root microbiomes from 20 cultivated angiosperms were compared. Surface-sterilized seeds from each species were sown in identical soil, and DNA was extracted from the plant roots after 7-8weeks. The bacterial (16S rRNA) and fungal (ITS) communities were then examined using Illumina MiSeq. The phylogenetic distances of host plants and assembly dissimilarities of bacterial microbiomes, but not of fungal ones, were significantly correlated, as were the topologies of the host plant phylogenetic tree and the community dissimilarity tree, thereby confirming the phylogenetic conservation of bacterial root microbiomes. Furthermore, host plant phylogeny mainly affected only a few specific bacterial lineages, including the Betaproteobacteria, Gammaproteobacteria, and Chloroflexi. Burkholderia (Betaproteobacteria) taxa were more abundant in monocots than in dicots, whereas Streptomyces (Actinobacteria) taxa were less abundant. These findings suggest that bacterial root microbiomes have significantly contributed to the functional divergence of angiosperms at higher taxonomic levels.

Draft Genome Sequences of Bacillus and Paenibacillus Species Isolated from Seeds of Citrullus lanata (Watermelon), Cucurbita moschata (Butternut Squash), and Cucurbita pepo L. var. pepo L. (Pumpkin).

Here, we announce the draft genome sequences of four endophytic bacilli isolated from surface-sterilized seeds of three cucurbit species, Bacillus sp. strains EKM417B and EKM420B (from Citrullus lanata [watermelon]) and EKM501B (from Cucurbita moschata [butternut squash]) and Paenibacillus sp. strain EKM301P (from Cucurbita pepo L. var. pepo L. [pumpkin]). These strains previously demonstrated biostimulant and biocontrol activities.

Draft Genome Sequences of Seven Strains of Paenibacillus spp. (Phylum Firmicutes) Inhabiting the Seeds of Cucumis melo L. (Cantaloupe) and Exhibiting Plant Probiotic Traits.

Here, we report the draft genome sequences of seven Paenibacillus sp. strains (EKM202P, EKM205P, EKM206P, EKM207P, EKM208P, EKM211P, and EKM212P) that were previously isolated from cultivated surface-sterilized seeds of Cucumis melo L. (cantaloupe). These candidate Paenibacillus plant probiotics displayed in vitro growth-promoting traits and suppressive activity against root-associated fungal/oomycete pathogens.

Diversity of the Seedborne Fungi and Pathogenicity of Fusarium Species Associated with Intercropped Soybean.

Maize/soybean relay strip intercropping has been widely practiced in Southwest China due to its high productivity and effective application of agricultural resources; however, several seedborne diseases such as seedling blight, pod and seed decay are frequently observed causing severe yield loss and low seed quality. So far, the population and pathogenicity of the seedborne fungi associated with intercropped soybean remain unexplored. In this study, seeds of 12 soybean cultivars screened for intercropping were collected from three growing regions in Sichuan Province of Southwest China, and the seedborne fungi were isolated from the surface-sterilized seeds. Based on sequence analysis of ribosomal DNA internal transcribed spacer (rDNA ITS), 148 isolates were identified into 13 fungal genera, among which Fusarium covered 55.0% as the biggest population followed by Colletotrichum. Furthermore, Fusarium isolates were classified into five distinct species comprising F. fujikuroi, F. proliferatum, F. verticillioides, F. asiaticum and F. incarnatum through sequence analysis of translation elongation factor 1 alpha (EF-1α) and DNA-directed RNA ploymerase II second largest subunit (RPB2). Among them, F. fujikuroi accounted for 51.22% (42/82) and was isolated from 91.7% (11/12) soybean varieties. Pathogenicity assay showed that five Fusarium species were able to infect the seeds of soybean cultivar “Nandou12” and caused water-soaked or rot symptoms, while F. fujikuroi and F. asiaticum had much higher aggressiveness than other species with significant reductions of seed fresh weight and germination percentage. Accordingly, this study indicates that Fusarium species are the dominant seedborne fungi in the intercropped soybean in Sichuan, China, and this provides some useful references for the effective management of seedborne fungal diseases as well as soybean resistance breeding in maize/soybean relay strip intercropping.

Seed inhabiting bacterial endophytes of finger millet (Eleusine coracana L.) promote seedling growth and development, and protect from fungal disease

The goal of this study was to evaluate the role of seed inhabiting bacterial endophytes on finger millet seedling development and protection from fungal infection. This study has shown that removal of endophytic bacteria from millet seeds compromised seedling health, however, re-inoculation of the same bacterial isolates restored its growth. A total of six endophytic bacteria were isolated from surface-sterilized seeds of finger millet and molecularly identified by 16S rDNA sequencing as Paenibacillus dendritiformis (EC1), Enterobacter hormaechei (EC2), Enterobacter cloacae (EC3), Bacillus safensis (EC4), Enterobacter hormaechei (EC5), and Enterobacter hormaechei (EC6). These isolated endophytes were tested for their plant growth-promoting activities. Enterobacter strains (EC2, EC3, EC5 and EC6) were found positive for IAA (Indole acetic acid) test and showed phosphate as well as potassium solubilization activities while siderophore production was shown by EC1 and EC4. Antifungal activity was also evaluated with isolate EC1 showing inhibition against all the tested phytopathogens. In seedling protection assay, bacterial endophytes significantly reduced the infection from Fusarium oxysporum. Re-inoculation experiment was carried out and it was found that bacterial strains EC1, EC4, and EC5 were most active in seedling development of finger millet, showing considerable improvement in root-shoot lengths, fresh weights and content of chlorophyll pigments. Endophytic bacterial colonization in the seedling roots was observed using fluorescent microscopy. This study reports the presence of endophytic bacteria inside seeds of finger millet having stimulatory effects on its growth and development.

Draft Genome Sequence of Bacillus sp. Strain EKM601B (Phylum Firmicutes), Living inside the Seeds of Luffa acutangula (Chinese Okra).

Presented here is the draft genome sequence of Bacillus sp. strain EKM601B, which contains 4,199,360 bp in 73 contigs. This candidate endophyte was isolated from surface-sterilized dry seeds of Luffa acutangula (Chinese okra) and demonstrated diverse plant-beneficial functions and antagonism against soilborne pathogens in vitro.

Occurrence of Macrophomina phaseolina Causing Root and Crown Rot on Alfalfa (Medicago sativa) in China

Alfalfa (Medicago sativa) is the widest grown and most important sown pasture crop in China and worldwide. Root and crown rot disease severely threatens alfalfa production, causing estimated annual yield losses of up to 20 to 40% worldwide (Fang et al. 2019). Field surveys in major alfalfa-growing areas in northwest China during summer (June to August) in 2017 to 2019 showed root and crown rot was common in fields established for more than 2 years with disease incidence ranging from 30 to 80%. Diseased plants were usually stunted and wilted; taproots and crowns exhibited red-brown to dark-brown discoloration in the cortex and/or vascular tissues. Frequently, lateral roots and stem tissues showed similar symptoms. Whole roots and crowns were decayed, with shoots wilted in severely affected plants. A total of 250 symptomatic plants were sampled randomly in Gansu Province from five alfalfa fields surveyed in 2018. Isolations were conducted by plating discolored root and crown tissues (40 pieces for each plant) sterilized with 1.25% sodium hypochlorite onto water agar. The plates were incubated at 22°C for 3 days, and then growing hyphae were subcultured onto potato dextrose agar (PDA). Twenty-one isolates from the fields in Zhangye, Gansu Province, were primarily identified as Macrophomina phaseolina based on cultural and morphological characteristics on PDA (Kaur et al. 2012; Sanchez et al. 2013). Five isolates picked randomly were purified by single-sclerotial isolation and maintained on 1/5 strength PDA at 4°C for further study. DNA of these isolates was extracted (Cenis 1992), and PCR amplifications of the internal transcribed spacer (ITS) region of rDNA (ITS1, 5.8S rDNA, and ITS2) were performed using primers ITS1 and ITS4. Sequences were then compared with the reference sequences in GenBank using BLAST search, showing sequence identity with M. phaseolina and the best matches at 98.47% (e.g., accession nos. MK408587 and KR012878). The ITS sequence of a representative isolate (Mp3) has been deposited in GenBank (accession no. MT192442). Pathogenicity tests of two isolates (Mp3 and Mp5) were conducted on 2-week-old alfalfa seedlings (cv. Longdong) with three to four leaves grown from surface-sterilized seeds. Seedlings were transplanted into pots (12 × 12 cm) containing pasteurized potting mix infested with mycelial-colonized millet seed inoculum of either isolate at a rate of 0.5% (w/w). Control seedlings were similarly transplanted into pots but with uninfested potting mix. Four seedlings were planted in a pot with 12 replicate pots for each treatment, and pots were watered every 2 days to free draining under controlled environment conditions (28°C, 12-h photoperiod, and 65% relative humidity). Three weeks postinoculation, all inoculated plants showed stunting symptoms, with discolored roots and crowns with a disease incidence of 100%, whereas the control plants showed no disease symptoms. To confirm Koch’s postulates, both isolates were reisolated from discolored root and crown tissues and again confirmed by morphological similarities and ITS sequencing as the inoculated isolates. Five purified isolates have been submitted to the Grassland Culture Collection Center, Lanzhou University (accession nos. LZU-MsR-Mp1 to Mp5). To our knowledge, this is the first report of M. phaseolina causing root and crown rot on alfalfa in China. M. phaseolina has been reported to cause root and crown rot on alfalfa in America, and on other legumes such as white clover in America, mungbean in India, and many other important crops worldwide (Kaur et al. 2012; Pandey et al. 2020; Pratt et al. 1998). Due to its broad host range and potential to be a significant pathogen in alfalfa-growing areas in China, further study on disease epidemic development is recommended.

Variability for Salt (NaCl) Tolerance of Six Ethiopian Pea (Pisum sativum var. abyssinicum) Landraces

The biotic approach to overcome salinity stress has recently received global attention. Hence, the identification of existing crop landraces that could enhance salt tolerance is of considerable value for agriculture. We examined six landraces of a pea to find out their relative tolerance levels under varying salt concentrations. Landraces’ seeds were obtained from four districts of Ethiopia at different altitudinal ranges (1379–2457 m a.s.l.). The landraces Wemberet 2017 and Wemberet 2-2017 from Ofla, Sirinka 2017 from Habru, Gedober 2017 from Gubalafto, Mekan 2017 and Tahtay-haya 2017 from Endamehoni were examined for salinity tolerance by growing them in four salt treatment levels (5 dS/m, 7 dS/m, 9 dS/m, and 15 dS/m). Distilled water (0 dS/m) was used as a control. Fifty surface-sterilized seeds per Petri-dish were sown for each salt treatment and the control. Growth features decreased with increasing salinity stress. Landraces Mekan 2017, Wemberet 2017, Sirinka 2017, and Gedober 2017 exhibited higher resistance (5–7 dS/m) to NaCl. Wemberet 2017 and Mekan 2017 landraces showed the best growth performance at 5 dS/m. Tahtay-haya was the most affected by salt stress.

Influence of Relative Humidity on Germination and Metal Accumulation in Vigna radiata Exposed to Metal-based Nanoparticles

The effect of single (0.1, 1, and 10 mg L−1) and binary mixtures (0.1 + 0.1, 1 + 1, and 10 + 10 mg L−1) of metal-based nanoparticles (CdO and CuO) on the germination of Vigna radiata was studied under two humidity ranges (70% and 80%). Filter paper-based tests were conducted. The surface-sterilized seeds were exposed to CdO and CuO under controlled environmental conditions (70% and 80% humidity at 35 °C). Germination rates were scored after 24 h and 48 h. The accumulation of metals was tested in seedlings after 48 h using inductively coupled plasma mass spectrometry. Compared with 70% humidity, the germination rate was higher under 80% humidity in all tested conditions. The germination rate of the CdO + CuO treatment was less than that of the single metal exposure under both humidities (70% and 80%) at 48 h. By two-way analysis of variance (ANOVA), we found that germination was greatly influenced by humidity. The accumulation of metal was higher in the CuO test than in the CdO test. Metal accumulation was concentration and humidity dependent, except for Cd accumulation in the CdO + CuO treatment. Here we show that the germination of seeds depends on the humidity and concentration of metal oxide nanoparticles. Understanding these strategies in seeds might help to avoid environmental and chemical stress and improve crop yield.