Short-term effects of soil solarization in suppressing Calonectria microsclerotia

Background and aims

Calonectria species have been reported as devastating pathogens mostly on horticultural and forest crops worldwide. Since these pathogens represent a serious threat for the nursery production, the aim of this study was to investigate on the short-term potential of soil solarization for eradicating Calonectria microsclerotia.

Methods

Twenty Calonectria isolates collected in Italy from different hosts and locations were identified by using DNA sequencing of β-tubulin. The effect of thermal regimes and innovative solarizing films on the soil survival of Calonectria microsclerotia was evaluated through time at different sampling periods in growth chamber and greenhouse experiments.

Results

Eleven and nine isolates were identified as Calonectria pauciramosa and Calonectria polizzii, respectively. No viable Calonectria inoculum was recovered after 12 days from all solarized plots inside ethylene-tetrafluoroethylene (ETFE) greenhouse and at 15-cm depth from solarized plots inside ethylene-vinyl-acetate (EVA) greenhouse. Under EVA cover, solarization killed C. pauciramosa microsclerotia within 9 and 17 d at 15- and 30-cm depths in soil, respectively, whereas no viable inoculum was retrieved within 6 and 12 days from solarized plots inside ETFE greenhouse.

Conclusions

This paper demonstrates that short-term soil solarization is effective for Calonectria microsclerotia suppression in nurseries, and shows that ETFE film as well as other innovative materials could improve this technique.     

Effect of Microsphaeropsis ochracea on Production of Sclerotia-borne and Airborne Conidia of Botrytis squamosa

Onion leaf blight, caused by Botrytis squamosa (Walker), is a destructive disease of onion. Conidia produced on overwintered sclerotia are the main source of initial inoculum, and those produced on lesions are responsible for secondary inoculum build-up. The biological control agent Microsphaeropsis ochracea (Carisse &; Bernier) was evaluated for its ability to control sclerotia-borne inoculum, to colonize onion leaves and reduce the production of conidia under field conditions. Colonisation by M. ochracea of onion leaves at different growth stages was monitored and its effect on B. squamosa sporulation on necrotic leaves was evaluated. Onion plots were treated with either Dithane® or with M. ochracea at 7–10-day intervals and according to inoculum production index (IPI). The concentration of airborne conidia and the number of lesions per leaf, on 20 plants per plot, were evaluated throughout the cropping season. The number of conidia produced per sclerotium treated with M. ochracea, was reduced by 75.5%. In the field, M. ochracea colonised only senescent or necrotic leaves and reduced the production of conidia on these leaves by an average of 82% as compared with untreated leaves. Best disease control was obtained by Dithane®, followed by M. ochracea applied at 7–10-day intervals. For the three years of the study, there were no significant differences in airborne concentrations of conidia in plots treated at 7–10-day intervals with Dithane® or M. ochracea. Fall application of M. ochracea could be used as a sanitation practice to reduce initial inoculum or as a part of an IPM program during the season.     

Identification of Cauliflower Cultivars that Differ in Susceptibility to Verticillium longisporum using Different Inoculation Methods

The response of 13 European cauliflower cultivars to Verticillium longisporum was evaluated using two greenhouse tests and one in vitro inoculation test. The greenhouse tests involved dipping roots of 3‐week‐old seedlings in a conidial suspension or inoculating the soil of 3‐week‐old seedlings with Verticillium microsclerotia. The in vitro test involved the inoculation of 9‐day‐old seedlings with Verticillium conidia. Useful disease parameters were the area under disease progress curve and plant growth reduction for the greenhouse tests and fresh weight reduction for the in vitro test. Significant correlations were found among the three inoculation methods. Irrespective of the inoculation method used, cultivar ‘Sernio’ was most resistant to V. longisporum, while ‘Minaret’ was the most susceptible cultivar. The pathogen could be re‐isolated from the hypocotyls and from the stem of ‘Minaret’ 4 and 49 days after inoculation respectively, whereas V. longisporum could never be re‐isolated from ‘Sernio’. These results suggest that the more resistant cauliflower cultivar ‘Sernio’ can suppress the ascent and the proliferation of V. longisporum into the plant.     

Effects of experimental warming on fungal disease progress in oilseed rape

Global warming will influence the growth and development of both crops and pathogens. The aims of this study were to investigate potential effects of future warming on oilseed rape growth and the epidemiology of the three economically important pathogens Verticillium longisporum, Sclerotinia sclerotiorum, and Leptosphaeria maculans (anamorph: Phoma lingam). We utilized climate chambers and a soil warming facility, where treatments represented regional warming scenarios for Lower Saxony, Germany, by 2050 and 2100, and compared results of both approaches on a thermal time scale by calculating degree‐days (dd) from day of sowing, December 1st and March 1st until sampling, the latter correlating best with disease progress. Regression analysis showed that plant growth and growth stages in spring responded almost linearly to increasing thermal time until 1000–1500 dd. Colonization of plant tissue by V. longisporum showed an exponential increase when exceeding 1300–1500 dd and reaching plant growth stage BBCH 74/75 (pod development). V. longisporum colonization of plants may be advanced, potentially leading to higher inoculum densities after harvest and increased economic importance of this pathogen under future warming. Sclerotia germination of S. sclerotiorum reached its maximum at 600–900 dd. Advance of these critical degree‐days may lead to earlier apothecia production, potentially advancing the infection window, whereas the future importance of S. sclerotiorum may remain constant. Severity of phoma crown canker increased linearly with increasing thermal time, but showed also large variation in response to the warming scenarios, suggesting that factors such as canopy microclimate in fall or leaf shedding over winter may play a bigger role for L. maculans infection and disease severity than higher soil temperatures. Thermal time was a suitable tool to combine and integrate data on biological responses to soil and air temperature increases from climate chamber and field experiments.     

Soil inoculum density of Verticillium dahliae and Verticillium wilt of olive in Lebanon

In the Mediterranean basin, Verticillium Wilt of Olive (VWO) is diffused throughout its range of cultivation, causing severe yield losses and tree mortality. The disease was reported in almost all the Mediterranean and Middle East countries, and in Lebanon it is of increasing significance also on many valuable crops. The disease has already been reported on potato, peach and almond in the Bekaa valley; however, to date no information is available about the incidence of VWO and the inoculum density of Verticillium dahliae microsclerotia in soil of the main agricultural areas of Lebanon. Results from the present investigations demonstrate a high V. dahliae frequency in soils (75.3%), coupled with a mean soil inoculum density of 17.0 MS g^?1, clearly indicating a great impact on the production of susceptible hosts in Lebanon, mainly in Bekaa region. Molecular method to assess the microsclerotia inoculum density in soil allowed the detection of a higher frequency of infested soils, as compared with the traditional plating, thus confirming its higher sensitivity. The overall Verticillium wilt prevalence in the inspected olive orchards was 46.2%, and the frequency of V. dahliae‐infected trees was 25.7%. The widespread presence of V. dahliae in all olive growing areas of Lebanon enforces the adoption of measures aimed at reducing the soil inoculum density before any new olive plantation, and the use of strong phytosanitary regulations to improve the certification schemes of propagating material.     

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.     

Liquid culturing of microsclerotia of Mycoleptodiscus terrestris,a potential biological control agent for the management of hydrilla

Mycoleptodiscus terrestris has potential as an inundative biological control agent for the management of hydrilla, one of the world’s worst aquatic weeds. Essential to producing a marketable bioherbicidal product was the development of liquid culture procedures that would yield propagules that maintained biocontrol efficacy. Since M. terrestris did not produce conidia in liquid culture, various nutritional conditions were evaluated as a means to produce high concentrations of stable fungal propagules such as microsclerotia. Evaluations of propagule formation and biomass yield were carried out in liquid culture media containing a basal salts solution amended with corn steep liquor powder or cottonseed meal combined with 4% or 6% glucose. Hyphal aggregation was observed by day 2, and by day 8 abundant melanized microsclerotia were present in the broth cultures. When applied as a liquid inoculum to hydrilla at rates of 0.1 and 0.2 ml/l, the microsclerotial matrix was capable of significantly reducing hydrilla shoot biomass by as much as 99%. Air-dried microsclerotia were capable of hyphal germination in 24 h and sporogenic germination in 72 h. These capabilities have significance for the use of microsclerotia of M. terrestris as the preferred inoculum for biocontrol purposes. Hyphae germinating from microsclerotia on hydrilla plant surfaces can establish initial infection sites followed several days later by secondary infections resulting from the development and release of spores from the surface of the microsclerotia. The capability of microsclerotia of M. terrestris to remain stable as a dry preparation and to germinate both hyphally and sporogenically upon rehydration enhances the potential of this fungus for use as a nonchemical, biological control agent for hydrilla.     

Differential resistance of oilseed rape cultivars (Brassica napus ssp. oleifera) to Verticillium longisporum infection is affected by rhizosphere colonisation with antagonistic bacteria, Serratia plymuthica and Pseudomonas chlororaphis

The effect of a seed treatment with the antagonistic bacteria Serratia plymuthica (strain HRO-C48) and/or Pseudomonas chlororaphis (strain MA 342) on the infection of oilseed rape with Verticillium longisporum was assessed with ten different cultivars. Soil was inoculated with microsclerotia and mycelium of a V. longisporum culture. Seeds were treated with rifampicin-resistant antagonistic bacteria at a rate of log₁₀ 6–7 cells per seed. Resistance against V. longisporum infection did not differ between cultivars and was generally low. A significant disease reduction recorded as area under disease progress curve (AUDPC) was obtained with both antagonistic rhizobacteria with no significant difference between the treatments. Percent of healthy plants was approximately 70% in all bacterial treatments. Significant differences were observed between the cultivars ranging from 46.5% (cultivar Titan) to 72.6% (Trabant). The combined use of both bacteria could not provide additional control effects. The bacterial density in the rhizosphere was not related to the control effect, but increased by log₁₀ 2 on infection with V. longisporum. Growth promotion effects were also not related to the control effect. At present, neither the application of chemical fungicides nor breeding for resistance against V. longisporum in oilseed rape can provide a solution for this increasingly problematic plant pathogen. The present results now open perspectives to control V. longisporum in oilseed rape by making use of cultivars, which express resistance against this pathogen on interaction with the antagonistic rhizobacteria S. plymuthica or P. chlororaphis.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.     

Genetic analysis of phenylpropanoid metabolites associated with resistance against Verticillium longisporum in Brassica napus

Verticillium longisporum is a major threat to production of oilseed rape (Brassica napus) in Europe. The aim of the study was to develop new markers and obtain insights into putative mechanisms and pathways involved in the resistance reaction. A genetic approach was used to identify quantitative trait loci (QTL) for V. longisporum resistance and metabolic traits potentially influencing resistance in a B. napus mapping population. Resistance to V. longisporum was mapped in a doubled haploid (DH) population from a cross between the partially resistant winter oilseed rape variety Express 617 and a resistant resynthesized B. napus line, R53. One major resistance QTL contributed by R53 was identified on chromosome C5, while a further, minor QTL contributed by Express 617 was detected on chromosome C1. Markers flanking the QTL also significantly correlated with V. longisporum resistance in four further DH populations derived from crosses between elite oilseed rape cultivars and other resynthesized B. napus lines originating from genetically and geographically diverse brassica A and C genome donors. The tightly-linked markers developed enable the combination of favorable alleles for novel resistance loci from resynthesized B. napus materials with existing resistance loci from commercial breeding lines. HPLC analysis of hypocotyls from infected DH lines revealed that concentrations of a number of phenylpropanoids were correlated with V. longisporum resistance. QTL for some of these phenylpropanoids were also found to co-localize with the QTL for V. longisporum resistance. Genes from the phenylpropanoid pathway are suggested as candidates for V. longisporum resistance.     

Effects of using different host plants and long-term fertilization systems on population sizes of infective arbuscular mycorrhizal fungi

The effect of cultivation of mycorrhizal and non-mycorrhizal plants and mineral fertilization on the arbuscular mycorrhizal fungal (AMF) community structure of maize (Zea mays L.) plants was studied. Soil samples were collected from two field experiments treated for 5 years with three fertilization systems (Control – no fertilization; Mineral – NPK fertilization; and Organic – Farmyard manure fertilization). Soil samples containing soil and root fragments of rapeseed (Brassica napus L., non-mycorrhizal plant) and wheat (Triticum aestivum L., mycorrhizal plant) collected from the field plots were used as native microbial inoculum sources to maize plants. Maize plants were sown in pots containing these inoculum sources for four months under glasshouse conditions. Colonization of wheat roots by AMF, AMF community structure, AMF diversity (Shannon’s index), AMF dominance (Simpson’s index) and growth of maize were investigated. Sixteen AMF species were identified from rhizosphere soil samples as different species of genera Acaulospora, Claroideoglomus, Dentiscutata, Funneliformis, Gigaspora, Quatunica, Racocetra, and Rhizoglomus. Maize plants grown in manure-fertilized soils had a distinct AMF community structure from plants either fertilized with mineral NPK-fertilizer or non-fertilized. The results also showed that inoculum from non-mycorrhizal plants combined with mineral fertilization decreased AMF diversity (Shannon’s index), AMF dominance (Simpson’s index) and growth of maize. Our findings suggest that non-mycorrhizal plants, such as B. napus, can negatively affect the presence and the effects of soil inoculation on maize growth. Also, our results highlight the importance of considering the long-term effect of rapeseed cultivation system on the reduction of population sizes of infective AMF, and its effect on succeeding annual crops.     

Impact of fungicides on Metarhizium anisopliae in the rhizosphere, bulk soil and in vitro

The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) is registered in the United States and The Netherlands for black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) control in container-grown ornamentals. These studies were conducted to determine the compatibility of M. anisopliae (F52) with a wide range of fungicides commonly applied to container-grown ornamentals for the management of soil-borne plant pathogens. The impact of fungicides on spore germination and mycelial growth were determined in vitro. In addition, M. anisopliae persistence in bulk and rhizosphere soil was determined 30 days following dual application of each fungicide at 7–28 days intervals as prescribed. A number of fungicides (thiophanate-methyl, dimethomorph, captan, triflumizole, triflozystrobin, pyraclostrobin, azoxystrobin) inhibited spore germination in vitro. A larger number of fungicides (fosetyl-AI, thiophanate-methyl, dimethomorph, captan, quintozene, triflumizole, fludioxanil, triflozystrobin, pyraclostrobin, fludiox-mefanox, iprodione, azoxystrobin, phosphorus acid/K-salts) inhibited mycelial growth in vitro. Only three fungicides (etridiazole, propamocard and mafanoxam) had no significant impact in vitro on spore germination or mycelial growth. While a number of fungicides had a detrimental impact in vitro, there was no impact on M. anisopliae populations in bulk soil following dual application of any fungicide. However, the fungicides captan and triflumizolet, which have a short reapplication interval, had a detrimental impact on M. anisopliae populations in the rhizosphere. As researchers develop rhizosphere competence as an alternative management strategy for black vine weevil, the fungicides captan and triflumizole should be avoided.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Effects of long-term chlorimuron-ethyl application on the diversity and antifungal activity of soil Pseudomonas spp. in a soybean field in Northeast China

The herbicide chlorimuron-ethyl has been applied widely for weed control in farmland, especially in soybean fields in China over the past decade, but the chronic effects of this herbicide on soil microorganisms, particularly Pseudomonas spp., is not well understood. Taking a continuously cropped soybean field in the town of Fuyuan—a soybean production base of Heilongjiang Province in Northeast China—as a case study, soil samples were collected from plots having received 0-, 5-, and 10-year applications of chlorimuron-ethyl (30 g active component of chlorimuron-ethyl/ha/year) to study the abundance and diversity of Pseudomonas spp. Meanwhile, an in vitro assay was used to examine the antifungal activities of isolated Pseudomonas spp. against soil-borne pathogens (Fusarium graminearum, Fusarium oxysporum, and Rhizoctonia solani) causing soybean root rot disease. The production of siderophore, hydrogen cyanide (HCN), and lytic enzymes (cellulase, pectinase, and chitinase) by Pseudomonas spp. was also investigated. With 5- and 10- year chlorimuron-ethyl application, the numbers of soil Pseudomonas spp. decreased from 121?×?10² CFU/g dry soil in the control to 40?×?10² CFU/g dry soil and 13?×?10² CFU/g dry soil, and the Shannon index values decreased from 6.23 to 3.71 and 1.73, respectively. The numbers of antifungal Pseudomonas spp. also decreased, and the proportions of Pseudomonas spp. with antifungal activities against the different test pathogens altered. All the antifungal Pseudomonas spp. could produce siderophore and HCN but not lytic enzymes. The results suggest that long-term application of chlorimuron-ethyl in continuously cropped soybean field had negative effects on the abundance and diversity of soil Pseudomonas spp., including species with different antifungal activities against pathogens. Siderophore and HCN rather than lytic enzymes formed the antifungal metabolites of Pseudomonas spp., and the number of antifungal Pseudomonas that can produce siderophore and HCN decreased markedly under application of chlorimuron-ethyl, especially after 10-year application.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Do arbuscular mycorrhizas or heterotrophic soil microbes contribute toward plant acquisition of a pulse of mineral phosphate?

Aims

We investigated the role of arbuscular mycorrhizal fungi (AMF) and heterotrophic soil microbes in the uptake of phosphorus (P) by Trifolium subterraneum from a pulse.

Methods

Plants were grown in sterilised pasture field soil with a realistic level of available P. There were five treatments, two of which involved AMF: 1) unsterilised field soil containing a community of AMF and heterotrophic organisms; 2) Scutellospora calospora inoculum (AMF); 3) microbes added as filtrate from the field soil; 4) microbes added as filtrate from the S. calospora inoculum; 5) no additions, i.e. sterilised field soil. After 11 weeks, plants were harvested: 1 day before (day 0), 1 day after (day 2) and 7 days after (day 8) the pulse of P (10 mg kg^?1).

Results

There was no difference among treatments in shoot and root dry weight, which increased from day 0 to day 8. At day 0, shoots and roots of plants in the colonised treatments had higher P and lower Mn concentrations. After the pulse, the rate of increase in P concentration in the shoots was slower for the colonised plants, and the root Mn concentration declined by up to 50 % by day 2.

Conclusions

Plants colonised by AMF had a lower rate of increase in shoot P concentration after a pulse, perhaps because intraradical hyphae accumulated P and thus reduced its transport to the shoots.     

Early Infection and Competition for Nodulation of Soybean by Bradyrhizobium japonicum 123 and 138

Interactions of soybean with Bradyrhizobium japonicum 123 (serogroup 123) and 138 (serogroup c1) were used to examine the relationship between early infection rates, competition for nodulation, and patterns of nodule occupancy. Both strains formed more infections in autoclaved soil (sterile soil) than in untreated soil (unsterile soil). Inoculation did not increase numbers of infection threads in unsterile soil-grown plants, where infection of proximal portions of primary roots was complete by 5 days after planting. Both strains infected and nodulated at similar rates in sterile soil. Nodules were always clustered on the upper root system, regardless of inoculation and soil treatment. Sixty-seven percent of the nodules of uninoculated plants grown in unsterile soil were occupied by rhizobia belonging to serogroups other than 123 or c1. Inoculation with strain 123 or 138 increased occupancy by that strain at the expense of residency by other rhizobia. Eighty-three percent of all nodules on plants dually inoculated with both strains in sterile soil contained strain 138. The corresponding value for plants inoculated in unsterile soil was 31%. Neither inoculum strain dominated occupancy of first-formed nodules in unsterile soil. It appears that north central Missouri soil may not have populations of highly competitive serogroup 123 and that early infection and nodulation rates do not contribute to the competitive success of strain 138.     

Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

Arbuscular mycorrhizal fungi enhance P uptake and alter plant morphology in the invasive plant Microstegium vimineum

Invasive plant species can interact with native soil microbes in ways that change how they use nutrients and allocate biomass. To examine whether Microstegium vimineum form symbiotic associations with arbuscular mycorrhizal fungi (AMF) and whether AMF mediate nutrient acquisition and growth of the plant, we conducted a field survey in Raleigh, NC and Hangzhou, China and two experiments in growth chambers. This is the first report that M. vimineum is mycorrhizal, with colonization rates of 47 and 21 % in its native and invaded range, respectively. In the growth chamber, addition of an AMF inoculum mixture significantly promoted M. vimineum biomass accumulation in both field and sterilized soils, particularly after 64 days of growth. Arbuscular mycorrhizal fungi also increased plant phosphorous (P) uptake but did not consistently affect total plant nitrogen (N) acquisition, leading to decreases in plant N:P ratios. More interestingly, AMF significantly altered plant morphology, increasing the number of stolons and aerial roots per individual (59 and 723 %), aerial roots per gram aboveground biomass (374 %) and aerial roots per stolon (404 %). Our results suggest that mycorrhizal enhancement of plant growth by stimulating tillering may serve as another mechanism by which M. vimineum can quickly take over new territory. Future studies on invasive plant-microbial interactions are needed to understand the mechanisms through which microbes contribute to the competitive ability of invasive plants.     

Ectomycorrhizal fungi increase the vitality of Norway spruce seedlings under the pressure of Heterobasidion root rot in vitro but may increase susceptibility to foliar necrotrophs

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.