J. Ole Becker

Identification of Fungal rDNA Associated with Soil Suppressiveness Against Heterodera schachtii Using Oligonucleotide Fingerprinting

ABSTRACT To understand the nature of a soil with suppressiveness against Heterodera schachtii, an rDNA analysis was used to identify fungi associated with H. schachtii cysts obtained from soils possessing various levels of suppressiveness. Because H. schachtii cysts isolated from these suppressive soils can transfer this beneficial property to nonsuppressive soils, analysis of the microorganisms associated with the cysts should lead to the identification of the causal organisms. Five soil treatments, generated by mixing different amounts of suppressive and fumigation-induced nonsuppressive soils, were infested with second-stage juveniles of H. schachtii and cropped with mustard-greens. Fungi were identified through an rDNA analysis termed oligonucleotide fingerprinting of ribosomal RNA genes (OFRG). Cysts obtained from soil mixtures consisting of 10 and 100% suppressive soil predominantly contained fungal rDNA with high sequence identity to Dactylella oviparasitica. The dominant fungal rDNA in the cysts isolated from the soil mixtures composed of 0.1 and 1% suppressive soil had high sequence identity to Fusarium oxysporum. Polymerase chain reaction (PCR) amplifications performed with sequence-selective primers corroborated the treatment-specific distribution of rDNA clones obtained by the OFRG analysis. When these sequence-selective PCR primers were used to examine H. schachtii cysts from biocidal soil treatments that produced various levels of suppressiveness, only the D. oviparasitica-like rDNA was consistently identified in the highly suppressive soils.

Efficacy of methyl iodide and synergy with chloropicrin for control of fungi

Efficacy of soil fumigation with methyl bromide and methyl iodide against Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Gliocladium virens, Phytophthora citricola, Phytophthora citrophthora, Pythium ultimum, Rhizoctonia solani and Verticillium dahliae was determined in laboratory experiments in closed fumigation chambers. Pythium ultimum was the most sensitive fungal species with EC 50 values for methyl bromide and methyl iodide of 15.5 and 8.6 μM, respectively. R solani was the least sensitive with EC 50 values of 253.4 and 161.4 μM for methyl bromide and methyl iodide, respectively. Relative potency ([methyl bromide]/[methyl iodide]) values ranged from 5.2 for P citricola to 1.5 for F oxysporum. Methyl iodide was 2.7 more efficacious than methyl bromide averaged over all fungal species. Methyl bromide/chloropicrin and methyl iodidelchloropicrin applied jointly were 2.2 and 2.8 times more efficacious, respectively, against F oxysporum than when the compounds were applied singly. Combining methyl bromide and methyl iodide with chloropicrin resulted in a significant synergistic increase in activity against F oxysporum.

Identification of root-knot nematode suppressive soils

In three of 12 soils obtained from agricultural fields in California, population density development of Meloidogyne incognita under susceptible tomato was significantly suppressed when compared to identical but methyl iodide (MI)-fumigated, M. incognita re-infested soils. When the 12 soils were infested with second-stage juveniles (J2) of M. incognita and the juveniles were extracted after 3 days, significantly fewer J2 were recovered from 9 of the 12 non-treated soils than from the MI-fumigated equivalents. In one of the 12 soils, infestation 3 weeks before planting resulted in lower nematode population densities than infestation at planting in both MI-fumigated and non-treated soil. The combination of infestation 3 weeks before planting with infestation at planting did not alter the occurrence or degree of root-knot nematode suppressiveness.

Suppression of the Plant-Parasitic Nematode Heterodera schachtii by the Fungus Dactylella oviparasitica

This study examined the role of the fungi Dactylella oviparasitica and Fusarium oxysporum in the beet-cyst nematode (Heterodera schachtii) suppressiveness exhibited by a southern Californian soil. In prior research, the abundance of D. oviparasitica rRNA genes positively correlated with high levels of suppressiveness, whereas the abundance of F. oxysporum rRNA genes positively correlated with minimal to moderate levels of suppressiveness. In this report, both fungi were added to fumigation-induced nonsuppressive soil, planted with Swiss chard, and infested with H. schachtii juveniles. After two nematode generations, D. oviparasitica strain 50 reduced the population densities of H. schachtii eggs and juveniles to those in the suppressive soil and H. schachtii cysts to levels lower than in the suppressive soil. F. oxysporum did not significantly reduce H. schachtii populations. These results suggest that D. oviparasitica strain 50 plays a major role in the suppression of H. schachtii population development in this southern Californian soil.

Bacterial rRNA Genes Associated with Soil Suppressiveness against the Plant-Parasitic Nematode Heterodera schachtii

ABSTRACT The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured α-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H. schachtii suppressiveness.

Surface application of ammonium thiosulfate fertilizer to reduce volatilization of 1,3-dichloropropene from soil

Atmospheric emission of the soil fumigant 1,3-dichloropropene (1,3-D) is of environmental concern because of its toxicity and carcinogenicity. Thiosulfate fertilizers have been found to rapidly transform 1,3-D in soil to non-volatile ions which are less toxic. We investigated the use of surface application of ammonium thiosulfate (ATS) for reducing 1,3-D volatilization. In packed soil columns, emission of 1,3-D applied by sub-surface injection decreased with increasing ATS application rate and the amount of water used for delivering ATS. When ATS was applied in 9 mm water at 64g m ˇ2 , total 1,3-D emission was reduced by 61%. The reduction was 89% when ATS was applied at 193 g m ˇ2 . Bioassays showed that ATS application did not affect the effectiveness of 1,3-D for controlling citrus nematodes. In field plots where a 1,3-D emulsified formulation was applied via sub-surface drip, surface spray of ATS reduced 1,3-D emissions by 50%, and by 71% when the surface was also covered with polyethylene film. ATS application had no effect on the efficacy of root-knot nematode control or tomato yields. These results suggest that surface application of thiosulfate fertilizers may be a feasible and effective strategy for minimizing 1,3-D emissions. # 2000 Society of Chemical Industry

Correlations between Root-Associated Microorganisms and Peach Replant Disease Symptoms in a California Soil

Background Replant disease often occurs when certain crops are “replanted” in a soil that had previously supported the same or similar plant species. This disease typically leads to reductions in plant growth, crop yields, and production duration, and its etiology remains ill-defined. The objective of this study was to identify microorganisms associated with peach replant disease symptoms at a field location in California, USA. Soil samples were subjected to treatments to create various levels of replant disease symptoms. Clonal peach seedlings were grown in the treated soils in greenhouse trials. After 6 weeks, plant growth parameters were measured, and both culture and culture-independent analyses were performed to identify root-associated bacteria, fungi and stramenopiles. Results A total of 295,785 bacterial operational taxonomic units (OTU) were identified by an Illumina-based, high throughput sequence analysis of rRNA genes. Among the 60 most abundant OTUs, 27 showed significant (P<0.05) negative correlation with peach shoot weights while 10 were positively correlated. Most of these OTUs belonged to the bacterial phylum Proteobacteria (96%), including the classes Gammaproteobacteria (44.4%), Betaproteobacteria (33.3%) and Alphaproteobacteria (22.2%), and the orders Pseudomonadales, Burkholderiales, Chromatiales, Rhodocyclales, and Sphingomonadales. The most abundant fungi were Trichoderma asperellum, Trichoderma virens, Fusarium oxysporum, Ceratocystis fimbriata and Fusarium solani. The most abundant stramenopiles were Pythium vexans, Pythium violae and an unidentified Aplanochytrium species. Validation experiments using sequence-selective quantitative PCR analyses identified negative and positive associations between P. vexans and Trichoderma spp. and peach shoot weights, respectively. Conclusions This study identified numerous microorganisms associated with peach replant symptoms, some of which have been previously identified while others represent new candidates. Subsequent Kochs postulates investigations will assess their possible roles in this replant disease.

Effect of soil physical factors on methyl iodide and methyl bromide

Production and importation of methyl bromide is scheduled to be banned by 2001. Methyl iodide was evaluated as a possible replacement soil fumigant. The effects of soil moisture, temperature, soil texture and fumigation time on the efficacy of methyl iodide for the control of two common weeds, Abutilon theophrasti and Lolium multiflorum, were characterized and compared with those of methyl bromide. The optimal soil moisture for methyl iodide to kill both weed species in sandy soils was 14% water content (w/w). Greater efficacy was obtained when the temperature during fumigation was above 20°C. Compared to methyl bromide, the efficacy of methyl iodide was more consistent in different soils. Time to 100% mortality of weeds was 24 h for methyl iodide fumigation and 36 h for methyl bromide when 200 μM of fumigant was used. On a molar basis methyl iodide was consistently more effective than methyl bromide across the range of environmental factors tested. In terms of application technology and spectrum of activity, methyl bromide can be directly replaced by methyl iodide.

Soil suppressiveness to Heterodera schachtii under different cropping sequences

Heterodera schachtii population densities were monitored in a H. schachtii -suppressive soil cropped in screenhouse experiments for two consecutive seasons with wheat, susceptible or resistant cultivars of either sugar beet or oilseed radish, or left fallow. Heterodera schachtii population densities under wheat and the resistant cultivars of sugar beet and oilseed radish did not differ significantly from the fallow treatment. Populations declined under all crops, with a reproductive factor between 0.08 and 0.57. In glasshouse experiments, introduced H. schachtii populations increased greatly on susceptible Swiss chard grown in previously wheat-monocultured soils, suggesting that significant loss of H. schachtii suppressiveness occurred during the monoculture. Following fallow, two H. schachtii -resistant or two H. schachtii -susceptible cultivars, introduced sugar beet cyst nematode populations remained small, suggesting that suppressiveness had been maintained. In a field trial with H. schachtii suppressive soil, cyst nematode population densities remained lower under wheat, resistant sugar beet, resistant radish and susceptible radish than under susceptible sugar beet.

Induction of Beet-Cyst Nematode Suppressiveness by the Fungi Dactylella oviparasitica and Fusarium oxysporum in Field Microplots

ABSTRACT The ability of Dactylella oviparasitica and Fusarium oxysporum to suppress Heterodera schachtii numbers was examined in field microplots. Fungi were individually added to fumigated field soil that was seeded with sugar beet. Four weeks later, soils were infested with H. schachtii second-stage juveniles (J2). At two harvests, 11 weeks and 19 weeks (1,469 and 2,547 degree days (base 8 degrees C), respectively) after nematode-infestation, H. schachtii cyst and egg numbers were assessed. At both time points, D. oviparasitica reduced H. schachtii population densities to those in the naturally suppressive soil, even when additional H. schachtii J2 were added to the microplots after the first harvest. Although F. oxy-sporum did not alter H. schachtii population densities after 11 weeks, significant reductions were detected after 19 weeks. The sustainability of the H. schachtii suppressiveness created by single applications of the fungi at the beginning of the microplot trials was further examined in a greenhouse study. Soil collected at the completion of the microplot trials was potted and seeded with sugar beet. Four weeks later, each pot was infested with H. schachtii J2. Approximately 16 weeks (1,389 degree days) after seeding, the D. oviparasitica-amended soil produced greater fresh root weights and considerably smaller nematode population densities than the nonamended control.