Illumina-based analysis of the rhizosphere microbial communities associated with healthy and wilted Lanzhou lily (<Emphasis Type="Italic">Lilium davidii</Emphasis> var. <Emphasis Type="Italic">unicolor</Emphasis>) plants grown in the field

Lanzhou lily (Liliumdavidii var. unicolor) is the best edible lily as well as a traditional medicinal plant in China. The microbes associated with plant roots play crucial roles in plant growth and health. However, little is known about the differences of rhizosphere microbes between healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants. The objective of this study was to compare the rhizosphere microbial community and functional diversity of healthy and wilted plants, and to identify potential biocontrol agents with significant effect. Paired end Illumina Mi-Seq sequencing of 16S rRNA and ITS gene amplicons was employed to study the bacterial and fungal communities in the rhizosphere soil of Lanzhou lily plants. BIOLOG technology was adopted to investigate the microbial functional diversity. Our results indicated that there were major differences in the rhizosphere microbial composition and functional diversity of wilted samples compared with healthy samples. Healthy Lanzhou lily plants exhibited lower rhizosphere-associated bacterial diversity than diseased plants, whereas fungi exhibited the opposite trend. The dominant phyla in both the healthy and wilted samples were Proteobacteria and Ascomycota, i.e., 34.45 and 64.01 %, respectively. The microbial functional diversity was suppressed in wilted soil samples. Besides Fusarium, the higher relative abundances of Rhizoctonia, Verticillium, Penicillium, and Ilyonectria (Neonectria) in the wilted samples suggest they may pathogenetic root rot fungi. The high relative abundances of Bacillus in Firmicutes in healthy samples may have significant roles as biological control agents against soilborne pathogens. This is the first study to find evidence of major differences between the microbial communities in the rhizospheric soil of healthy and wilted Lanzhou lily, which may be linked to the health status of plants.     

Combined use of biocontrol agents and zeolite as a management strategy against <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Verticillium</Emphasis> wilt

Vascular wilt pathogens, like Fusarium oxysporum and Verticillium dahliae, cause heavy economic loses to a range of crops. The lack of chemical control intensifies the problem. In the present study, the initial in vitro activity of 134 bacterial isolates, originating from various stages of the composting process of cotton residues, against F. oxysporum f. sp. melonis (FOM) and V. dahliae was evaluated. The most efficient strains, named SP10 and C20 M, belong to Bacillus sp. Both strains significantly reduced Fusarium and Vertilicillium wilt in melon and aubergine respectively. Furthermore, zeolite was tested alone or in combination with SP10 against V. dahliae and FOM. It was shown that the combination of zeolite and SP10 in the transplant soil plug was the most disease suppressive treatment. Interestingly the single application of zeolite was also plant-protective. The positive effect of zeolite on plant health could be linked with the recorded up-regulation of plant defense genes.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

QTL-seq and marker development for resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in cultivated watermelon

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

Fungal microbiota from rain water and pathogenicity of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.     

Impact of high temperature on sucrose translocation,sugar content and inulin yield in <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. var. <Emphasis Type="Italic">sativum</Emphasis>

Aims

Dianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.

Methods

ipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.

Results

Inoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high titres.

Conclusion

Klebsiella SGM 81 effectively improves root traits of D. caryophyllus in a dose dependent manner, likely through tryptophan dependent IAA production in the rhizoplane and potentially within the intercellular spaces of root tissue. Under optimal plant growth promoting conditions in non-sterile soil, the high total microbial titre in the rhizosphere supports a mutualistic relationship between Klebsiella SGM 81 and carnation that potentially extends to the wider rhizosphere microbiota.

     

Incidence of <Emphasis Type="Italic">Fusarium</Emphasis> spp. on the invasive <Emphasis Type="Italic">Spartina alterniflora</Emphasis> on Chongming Island,Shanghai, China

Fusarium palustre is an endophyte/pathogen of Spartina alterniflora, a saltmarsh grass native to North America that has been associated in the USA with a saltmarsh decline known as Sudden Vegetation Dieback (SVD). Since the intentional introduction of S. alterniflora to stabilize mud flats on Chongming Island, Shanghai, China, S. alterniflora has become invasive, but shows no symptoms of dieback even though F. palustre can be isolated from the plant. When declining S. alterniflora from SVD sites in the northeastern USA were assayed for Fusarium species, an average of 8 % of tissues sampled gave rise to a species of Fusarium of these, 64 % were F. palustre and 16 % were F. incarnatum, a nonpathogenic species. To determine if low densities of F. palustre could explain the lack of dieback symptoms on S. alterniflora from Chongming Island, we assessed the incidence and distribution of Fusarium spp. on S. alterniflora from 12 sites on Chongming Island. On average, 26 % of the stem and root tissues sampled were colonized by a Fusarium species. Of 196 isolates recovered from S. alterniflora, 44 % were F. incarnatum and 41 % were F. palustre. Species determinations were confirmed for a subset of these isolates using a phylogenetic analysis of partial sequences of the translation elongation factor (tef) gene. The observation that Fusarium incidence on S. alterniflora was much greater on Chongming Island than in the USA survey raises the question as to why S. alterniflora on Chongming Island is showing no dieback. Other factors, such as predator release, enhanced nutritional, edaphic and/or other unidentified environmental constraints on Chongming Island may afford S. alterniflora protection from dieback.     

生物有机肥对土壤微生物群落结构变化及西瓜枯萎病的调控

【目的】研究施用生物有机肥处理土壤后对西瓜枯萎病发生的影响,明确可能与西瓜枯萎病发病密切相关的土壤微生物群落结构。【方法】设置2组不同年份材料对比,依次为施用生物有机肥后2016年发病期(CK2016、T12016)和2017年发病期(CK2017、T12017)。基于16S r RNA序列测定,利用Illumina Miseq高通量测序平台对施用生物有机肥后不同年份的土壤微生物群落组成和差异进行测序分析。【结果】试验结果发现,不同年份的土壤微生物群落中alpha多样性指数并无明显的差异,但是施用生物有机肥后的土壤相比对照土壤中细菌群落多样性略有增高。不同年份土壤细菌群落结构在门水平上差异不显著,其中变形菌门和硬壁菌门是构成这两个年份土壤细菌的重要组成部分,同时也是比较稳定的微生物类群。在属水平上分析发现主要动态变化菌属为芽孢杆菌属、肠球菌属、乳球菌属及水恒杆菌属。通过Spearman分析发现它们与西瓜枯萎病发生率的关系均为负相关。【结论】施用生物有机肥可帮助西瓜连作土壤的生态修复,对枯萎病防治起到一定的作用。通过对施用生物有机肥的不同年份土壤微生物种群结构动态变化及对枯萎病发生率呈正负相关的微生物菌属的分析研究,为如何利用调节土壤微生物群落结构来防治西瓜枯萎病提供了新的思路。     

Biocontrol of the toxigenic plant pathogen <Emphasis Type="Italic">Fusarium culmorum</Emphasis> by soil fauna in an agroecosystem

In 2011 and 2013, a field experiment was conducted in a winter wheat field at Adenstedt (northern Germany) to investigate biocontrol and interaction effects of important members of the soil food web (Lumbricus terrestris, Annelida; Folsomia candida, Collembola and Aphelenchoides saprophilus, Nematoda) on the phytopathogenic fungus Fusarium culmorum in wheat straw. Therefore, soil fauna was introduced in mesocosms in defined numbers and combinations and exposed to either Fusarium-infected or non-infected wheat straw. L. terrestris was introduced in all faunal treatments and combined either with F. candida or A. saprophilus or both. Mesocosms filled with a Luvisol soil, a cover of different types of wheat straw and respective combinations of faunal species were established outdoors in the topsoil of a winter wheat field after harvest of the crop. After a time span of 4 and 8 weeks, the degree of wheat straw coverage of mesocosms was quantified to assess its attractiveness for the soil fauna. The content of Fusarium biomass in residual wheat straw and soil was determined using a double-antibody sandwich (DAS)-ELISA method. In both experimental years, the infected wheat straw was incorporated more efficiently into the soil than the non-infected control straw due to the presence of L. terrestris in all faunal treatments than the non-infected control straw. In addition, Fusarium biomass was reduced significantly in all treatments after 4 weeks (2011: 95–99%; 2013:15–54%), whereupon the decline of fungal biomass was higher in faunal treatments than in non-faunal treatments and differed significantly from them. In 2011, Fusarium biomass of the faunal treatments was below the quantification limit after 8 weeks. In 2013, a decline of Fusarium biomass was observed, but the highest content of Fusarium biomass was still found in the non-faunal treatments after 8 weeks. In the soil of all treatments, Fusarium biomass was below the quantification limit. The earthworm species L. terrestris revealed a considerable potential as an effective biocontrol agent contributing to a sustainable control of a Fusarium plant pathogen in wheat straw, thus reducing the infection risk for specific plant diseases in arable fields.     

QTL mapping for resistance to Ceratocystis wilt in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.     

Temperature and soil microorganisms interact to affect <Emphasis Type="Italic">Dodonaea viscosa</Emphasis> growth on mountainsides

There is considerable interest in understanding the drivers of plant growth in the context of climate change. Soil microorganisms play an important role in affecting plant growth and functional traits. However, the role of interaction between soil microbes and temperature in affecting plant growth and functional traits remains unclear. The objective of this research was to investigate the effects of soil microbes, temperature, and their interaction on the growth and functional traits of Dodonaea viscosa in a mountain in Yuanmou county, southwest China. The experiment was conducted in climate chambers with a factorial design of three soil microbial communities (inoculated rhizosphere microbes from high elevation, inoculated rhizosphere microbes from low elevation, and autoclaved control) and two temperature conditions (colder and warmer). D. viscosa planted in inoculated rhizosphere microbes from both high and low elevations produced more total biomass with a lower root–shoot allometric exponent, and accumulated significantly more N and P nutrients than those in an autoclaved control, with no significant differences between the two microbial inoculations. Thus, rhizosphere soil microorganisms had positive effects on D. viscosa growth. However, the effect of the microbes on plant growth strongly depended on temperature. Warming had a positive effect on D. viscosa growth in inoculated rhizosphere microbe treatments, while the positive effect disappeared in the autoclaved control treatment. Our results indicate that temperature and soil microorganisms interact to affect D. viscosa growth. As the climate changes in the future in the studied region, the growth of D. viscosa may be greatly affected both directly and indirectly through the temperature–soil microbe interaction.     

Bacterial community structure associated with the rhizosphere soils and roots of <Emphasis Type="Italic">Stellera chamaejasme</Emphasis> L. along a Tibetan elevation gradient

The effect of altitude on the composition and diversity of microbial communities have attracted highly attention recently but is still poorly understood. We used 16S rRNA gene clone library analyses to characterize the bacterial communities from the rhizosphere and roots of Stellera chamaejasme in the Tibetan Plateau. Our results revealed that Actinobacteria and Proteobacteria were dominant bacteria in this medicinal plant in the rhizosphere and root communities. The Shannon diversity index showed that the bacterial diversity of rhizosphere follows a small saddle pattern, while the roots possesses of a hump-backed trend. Significant differences in the composition of bacterial communities between rhizosphere and roots were detected based on multiple comparisons analysis. The community of Actinobacteria was found to be significantly negative correlated with soil available P (p?<?0.01), while the phylum of Proteobacteria showed a positive relationship with available P (p?<?0.05). Moreover, redundancy analysis indicated that soil phosphorus, pH, latitude, elevation and potassium positively correlated with bacterial communities associated with rhizosphere soils. Taken together, we provide evidence that bacterial communities associated with S. chamaejasme exhibited some certain elevational pattern, and bacterial communities of rhizosphere soil were regulated by environmental characteristics along elevational gradients in this alpine ecosystem.     

Dispersal of <Emphasis Type="Italic">Fusarium</Emphasis> spp. by rainwater and pathogenicity on four plant species

Research focused on the occurrence of Fusarium spp. in atmospheric dust or rainwater is not common. Preliminary studies with four sampling dates in 2007 revealed that several species of Fusarium may also be conveyed by rainwater. In order to determine the regular presence of Fusarium spp. in rainfall water, samples were systematically collected for a year (from October 2009 to October 2010) in three points on the Mediterranean coast of the province of Granada (Spain) 10-km distance between them. Throughout the year of sampling, a total of 179 rainwater samples were collected during every significant rainfall event. Eight different Fusarium species were isolated from the rainwater samples: F. oxysporum (32 %), F. proliferatum (26 %) and F. equiseti (20 %) coincide with previous studies, while F. dimerum (3 %), F. semitectum (4.7 %), F. solani (8 %), F. avenaceum (0.5 %) and F. chlamydosporum (3.7 %) were isolated for the first time from rainwater. Results were consistent with previous surveys conducted 100 km away from the sampling sites. Inoculation of 39 different isolates from five different Fusarium species showed pathogenicity on plants. Disease severity differed depending on the inoculated plant species, which means that rain water can be an effective vector to transport new pathogens into new cultivated areas. This work reveals some epidemiological aspects of Fusarium genus in natural environments. Some of the isolated Fusarium spp. are potential mycotoxin producers, such as zearalenone, fumonisin, moniliformin or nivalenol.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.