T.C. Caesar-TonThat

Soil aggregate stabilization by a saprophytic lignin-decomposing basidiomycete fungus. I. Microbiological aspects

Abstractu2002We studied the effects of a saprophytic lignin-decomposing basidiomycete isolated from plant litter on soil aggregation and stabilization. The basidiomycete produced large quantities of extracellular materials that bind soil particles into aggregates. These binding agents are water-insoluble and heat-resistant. Water stability of aggregates amended with the fungus and the degrees of biodegradation of the binding agents by native soil microorganisms were determined by the wet-sieving method. The data demonstrated that aggregates supplemented with a source of C (millet or lentil straw) were much more water-stable and resisted microbial decomposition longer than when they were prepared with fungal homogenates alone. Moreover, retrieval of fungal-amended aggregates supplemented with millet during the first 4u2009weeks of incubation in natural soil exhibited more large aggregate fractions (>2u2009mm) than the ones supplemented with lentil straw. The possible relationship of the role of basidiomycetes in litter decomposition and soil aggregation is discussed.

Generation of antibodies for soil aggregating basidiomycete detection as an early indicator of trends in soil quality

Abstract Polyclonal antisera were raised against cell walls of a soil aggregating basidiomycete isolated in eastern Montana (isolate BB1) which has been identified as a member of the russuloid clade using molecular genetic techniques. In cross-reactivity tests using quantitative enzyme-linked immunosorbent assay (ELISA), polyclonal antisera to BB1 cross-reacted significantly with fungal species representative of the russuloid clade and little or no reactivity was observed with fungal species of the polyporoid, euagaric, hymenochaetoid, bolete and gomphoid–phalloid clades of the Homobasidiomycetes. These results suggested that the cell walls of fungal species from the russuloid clade share common antigenic binding sites that are recognizable by the polyclonal antibodies and that these sites were not found or were present in small amounts in fungal species from the other clades. Experiments on the water stability of artificial aggregates amended with fungal species representative of these six homobasidiomycete clades indicated that many species of the russuloid clade were very efficient soil stabilizers. Fungal species from the other clades vary in their ability to aggregate soil particles but cross-react weakly with the antibodies. ELISA was used on water stable aggregates (WSA) from soil samples of three dryland locations under conventional tillage, no tillage and fallow management practices and on WSA from soil samples from grass barrier strips that were undisturbed for 30 years. Greater antigenic response was observed from WSA of undisturbed soils compared to cultivated soils and from WSA of soils under no till compared to till or fallow management practices. These results suggested that specific soil aggregating russuloids in WSA are sensitive to soil disturbance such as tillage. To our knowledge, this study is the first report of the detection and quantification of a taxonomic group of specific soil aggregating Homobasidiomycetes in dryland soils under diverse agricultural systems.

Long-term tillage and cropping effects on microbiological properties associated with aggregation in a semi-arid soil

Little is known about the long-term tillage and cropping management effects on the microbiologically derived factors that influence macroaggregates in semi-arid soil. We tested the hypothesis that differences in macro-aggregation are due to changes in soil structure related to management treatment-induced microbiological changes. In an experiment, microbiological factors consisting of aggregate stability, glomalin, russuloid basidiomycete fungi, uronic acids, total organic C (TOC), and total N (TN) were quantified in macroaggregate-size classes ranging from 4.75 to 0.25xa0mm, collected at 0–5xa0cm depth for the following treatments: (1) 12th year of fallow phase after 11xa0years of conventional- and no-tilled spring wheat-fallow (CTF and NTF), (2) 12th year of lentil phase after 11xa0years of conventional- and no-tilled spring wheat-lentil (CTL and NTL), (3) 12xa0years no-tilled continuous spring wheat (NTCW), and (4) 16xa0years uncultivated pasture (P) used as a baseline treatment. Immunoreactive easily extractable glomalin concentration was five to six times greater under P, NTCW, or NTL in the 2.00–1.00- and 1.00–0.50-mm macroaggregate-size classes than the other treatments and these results corroborated well with the results from aggregate stability assays. Russuloid basidiomycetes were highest in all NTCW macroaggregate-size classes, suggesting that annual input of lignin-containing wheat residues may influence the growth and survival of these fungi. Uronic acid amounts were highest in P but did not differ among the other treatments. In all macroaggregate-size classes, TOC content was greater in NTCW compared to CTF, and TN was about three times higher in NTL than NTF or CTF. In conclusion, 12xa0years of NTCW management in semi-arid soil has resulted in higher macroaggregate stability, glomalin concentration, russuloid basidiomycete populations, and TOC in macroaggregates compared to alternate-year fallow. Lentil can be used to replace fallow in dryland wheat rotation under no-till to enhance TN content and improve soil macro-aggregation.

Dryland Soil Chemical Properties and Crop Yields Affected By Long-Term Tillage and Cropping Sequence

Information on the effect of long-term management on soil nutrients and chemical properties is scanty. We examined the 30-year effect of tillage frequency and cropping sequence combination on dryland soil Olsen-P, K, Ca, Mg, Na, SO4–S, and Zn concentrations, pH, electrical conductivity (EC), and cation exchange capacity (CEC) at the 0–120xa0cm depth and annualized crop yield in the northern Great Plains, USA. Treatments were no-till continuous spring wheat (Triticum aestivum L.) (NTCW), spring till continuous spring wheat (STCW), fall and spring till continuous spring wheat (FSTCW), fall and spring till spring wheat–barley (Hordeum vulgare L., 1984–1999) followed by spring wheat–pea (Pisum sativum L., 2000–2013) (FSTW-B/P), and spring till spring wheat-fallow (STW-F, traditional system). At 0–7.5xa0cm, P, K, Zn, Na, and CEC were 23–60% were greater, but pH, buffer pH, and Ca were 6–31% lower in NTCW, STCW, and FSTW–B/P than STW-F. At 7.5–15xa0cm, K was 23–52% greater, but pH, buffer pH, and Mg were 3–21% lower in NTCW, STCW, FSTCW, FSTW–B/P than STW-F. At 60–120xa0cm, soil chemical properties varied with treatments. Annualized crop yield was 23–30% lower in STW-F than the other treatments. Continuous N fertilization probably reduced soil pH, Ca, and Mg, but greater crop residue returned to the soil increased P, K, Na, Zn, and CEC in NTCW and STCW compared to STW-F. Reduced tillage with continuous cropping may be adopted for maintaining long-term soil fertility and crop yields compared with the traditional system.

Direct Polymerase Chain Reaction-Based Detection of Cercospora beticola in Field Soils

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

First Report of Rhizoctonia spp. Causing a Root Rot of the Invasive Rangeland Weed Lepidium draba in North America

The exotic, invasive perennial rangeland weed Lepidium draba spreads rapidly and reduces native species diversity. The extensive root system of L. draba constitutes 76% of its biomass (4). Thus, searches have been done for biocontrol agents that target root tissue or that may interact with a weevil, Ceutorhynchus assimilis, that causes galls in the crown area of L. draba. An association of Rhizoctonia spp. with root tissue of plants galled by the weevil has been documented in Europe (3). The possible presence of soilborne pathogens similar to those found in the native range has been the subject of L. draba surveys in the United States. One such survey in 2008 detected a few plants with reddened and chlorotic foliage in a stand near Shepherd, MT. Such symptoms typically indicate the occurrence of soilborne diseases on L. draba in the native range of the weed (2). The site had shown a gradual increase in the range of detectable pathogens beginning with foliar pathogens in 1997. In 2010, at the Shepherd site, L. draba plants with similar (but more severe) symptoms to those seen in 2008 were noted in a different area of the stand. Excavation of the roots in both years revealed brown, sunken crown and root cankers. Pieces of root tissue were excised from the lesions and plated on acidified PDA and Ko and Hora medium. A non-sporulating fungus was isolated from three plants. Colonies of the isolates on PDA were typical of known Rhizoctonia spp. The 2010 isolates were determined to be multinucleate using DAPI and were paired with 14 tester (including subgroups) isolates of AG-1 to AG-4 on water agar. Anastomosis was observed between the multinucleate isolates and the AG-2-1 tester isolate. Sequence analysis of ITS of the rDNA of a multinucleate isolate (GenBank KJ545577) indicated 99% similarity with an accession of R. solani AG 2-1 (AB547381). The 2008 isolates were binucleate. A binucleate isolate, KJ545578, had 100% similarity with an isolate of Rhizoctonia spp. AG-A (AY927356). Pathogenicity tests consisted of planting 6-week-old seedlings of L. draba, one per pot, in ten 85-cm-diameter pots of pasteurized soil mix infested with Rhizoctonia-colonized barley grain that had been dried and milled. An inoculum level of ~8 CFU/g (1) of air-dried soil was established by most probable number calculations from fourfold dilutions of infested soil. Controls were the same number of plants in pasteurized potting mix. Results were recorded after 3 months in a greenhouse at 20-25°C. The test was repeated. Typically, R. solani caused mortality of six to eight plants, from which it was re-isolated, whereas binuclate isolates caused stunting and lower dry weight of L. draba. Control plants remained asymptomatic. This is the first report of R. solani and binucleate Rhizoctonia spp. on L. draba in North America. References: (1) A. J. Caesar et al. Plant Dis. 93:1350, 2009. (2) A. J. Caesar et al. Biol. Control 52:140, 2010. (3) A. J. Caesar et al. Plant Dis. 96:145, 2011. (4) R. F. Miller et al. Agronomy J. 86:487, 1994.

First Report of Spot Form Net Blotch Caused by Pyrenophora teres f. maculata on Barley in the Mon-Dak Area of the United States

Pyrenophora teres Drechs. causes net blotch of barley, a common foliar disease in cultivation zones around the world. The disease occurs in two forms, namely a net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. As in other parts of the northern Great Plains, in the Mon-Dak area (western North Dakota and eastern Montana), NFNB is prevalent. SFNB was first reported in western Montana in 1983 (1) and more recently in eastern North Dakota in 2010 (3) but not in the Mon-Dak area. In the summer of 2011, unusual spot lesions that were surrounded by necrosis or chlorosis were observed on different barley cultivars in fields at Williston, ND, Nesson Valley, ND, and Sidney, MT areas. Diseased leaves from various barley cvs. from the three locations were transferred to water agar and incubated at room temperature for 24 h to induce sporulation. Morphological examination of conidia (45 to 169 × 15 to 21 μm) did not show significant differences from a known isolate of P. teres f. teres 0-1 (provided by Tim Friesen, ARS, Fargo, ND). For pathogenicity testing, six 14-day-old plants of barley cv. Tradition were sprayed until runoff with a 2,000 spore/ml suspension of two isolates from each location and the control P. teres f. maculata isolate DEN2.6 (provided by Tim Friesen). Plants were incubated first in a lighted humidity chamber for 24 h and then in a greenhouse for 7 days at 21°C. Regardless of inoculum source, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Fungi isolated from symptomatic leaves were identified as P. teres and the morphology of the conidia was undistinguishable from those of P. teres f. teres. All control plants which were sprayed with sterile distilled water were symptomless. The pathogenicity test was repeated. Rapid PCR detection and amplicon sequencing (2) of the internal transcribed spacer (ITS) region of ribosomal genes was performed on field and pathogenicity test leaf lesion samples to confirm the presence of P. teres f. maculata. DNA templates were prepared using the Extract-N-Amp Plant PCR Kits (Sigma Chemical Co., St. Louis, MO) and subjected to PCR using ITS1 and ITS4 primers. Amplicons were then purified and sequenced. The 585-bp nucleotide sequences of P. teres f. maculata from Mon-Dak area were submitted to GenBank under accession nos. PtmNES1 (JX187587), PtmSDY1 (JX187588), PtmSDY2 (JX187589), and PtmWIL1 (JX187590). The sequences from the four locations shared 100% similarity and also with P. teres f. maculata (EF452471) from GenBank while showing 10 nucleotide differences (99% similarity) with P. teres f. teres (EF452472).The results represent first report of SFNB in the Mon-Dak. Barley is one of the most important crops in the area. Resistance of the NFNB and SFNB of barley are controlled by different genes (4). Based on this report, SFNB therefore have to be considered in selection of barley cultivars for cultivation in the area. References: (1) H. E Bockelman et al. Plant Dis. 67:696, 1983. (2) R. T. Lartey et al. J. Sugar Beet Res. 40:1, 2003. (3) Z. H. Liu and T. L. Friesen. Plant Dis. 94:480, 2010. (4) O. M. Manninen et al. Genome. 46:1564, 2006.

First Report of a Root and Crown Disease of the Invasive Weed Lepidium draba Caused by Phoma macrostoma

The exotic rangeland perennial Lepidium draba occurs as a noxious weed in 22 states, mostly in the western United States. Because chemical control measures against this invasive perennial, a member of the Brassicaceae, have not achieved adequate results, biological control is being pursued. While inventories of arthropods that feed on L. draba have been established, little is known of soilborne pathogens for possible use as biological control agents. To address this deficiency, we have surveyed for diseases of L. draba in the United States and Eurasia to identify and test potential biocontrol agents. In intensive surveys for soilborne diseases in a single infestation that is >20 years old in a cattle pasture in south-central Montana, several chlorotic, stunted plants were noted. Roots of chlorotic plants that exhibited elongated fissures from which other soilborne fungi were isolated also had numerous prominent pycnidia embedded in the crown tissue above the lesions. Examination with a dissecting microscope revealed large ostioles made evident by the wide concave inversions in the short necks of the pycnidia. Culture of root tissue on potato dextrose agar resulted in whitish, becoming pale gray colonies, with a dull peach-to-reddish tinge at the margins, with abundant single pycnidia. Conidia in vitro were mainly unicellular, variable shape, subglobose to ellipsoidal, with several guttules averaging 6 × 2.5 μm. These morphological traits are characteristic of Phoma macrostoma, which is regarded as a weak or wound pathogen. The internal transcribed spacer region of rDNA was amplified using primers ITS1 and ITS4 and sequenced. BLAST analysis of the 575-bp fragment showed a 100% homology with the sequence of an isolate of P. macrostoma that has been investigated extensively for commercialization as a biological control agent of various agricultural weeds (1), including wild mustard (GenBank No. DQ474091). The nucleotide sequence has been assigned GenBank No. HM755951. Pathogenicity tests consisted of making four 1.4-mm-diameter holes in five NaOCl (0.1%)-sterilized root sections of L. draba and pipetting ~50 to 100 μl of a 106 CFU/ml conidial suspension into the incisions, incubating the inoculated roots at 20 to 25°C overnight and planting the root sections, one per pot, in an artificial greenhouse potting mix and placing the pots in the greenhouse at 20 to 25°C. Controls were five root sections that were treated similarly except that sterile water was injected. The experiment was repeated. After 10 days, shoots that grew from inoculated roots were chlorotic and shorter than those produced from control roots. P. macrostoma was isolated from tissue of inoculated roots that became blackened distal to the inoculation points. To examine the host range of P. macrostoma on other brassica species, crowns of 2-week-old seedlings of radish, broccoli, cauliflower, broccoli raab, turnip, kohlrabi, cabbage, Chinese cabbage, mustard greens, and canola were injected with 0.5 ml of a 106 CFU/ml conidial suspension. Plants were grown in the greenhouse at 20 to 25°C for 4 weeks after inoculation and examined for symptoms. The experiment was repeated twice. Blackened root tissue with slight chlorosis occurred only on roots of radish and crowns of broccoli, from which P. macrostoma was reisolated. To our knowledge, this the first report of a disease of L. draba caused by P. macrostoma. Reference: (1) K. L. Bailey et al. U.S. Patent Application Serial No. 60/294,475, Filed May 20, 2001.

First Report of Anthracnose Stem Canker of the Invasive Perennial Weed Lepidium draba Caused by Colletotrichum higginsianum in Europe

Exotic perennial Lepidium draba, native to Eurasia, is an invasive weed in dense stands in rangelands and disturbed areas in several states of the western United States and an agricultural weed in the prairie provinces of Canada. To determine strategies, such as a potential multipathogen strategy (1), for biological control of the weed, surveys that included the native range were conducted in spring 2009 to detect diseases that occur on this weed. Several stunted and chlorotic plants were found scattered throughout a stand of L. draba growing in a vacant lot near Riddes, Switzerland (46°0822.99″N, 7°919.02″E): ( http://maps.google.com/maps?source=earth&ll=46.13983490,7.15503250&layer= c&cbll=46.13983490,7.15503250&cbp=1,360,,0,5 ). Affected plants had reddish brown cankers on the lower stems, usually elongated and irregular in shape and slightly sunken. Insect injury was associated with the cankers. Symptoms often occurred on plants that were also infected with Rhizoctonia solani. After surface disinfestation with 0.1% sodium hypochlorite, tissue adjacent to and including lesions were plated on acidified potato dextrose agar and incubated at 20 to 25°C for 1 week. Zonate, dark gray colonies with sparse mycelia resulted that exhibited abundant, faintly pink spore masses with numerous dense clusters of black setae. Spores were single celled, hyaline, cylindrical to oval shaped, and 13.5 to 19.5 × 4 to 5.5 μm. Setae were 1- to 3-septate and 20 to 42 × 3 to 5 μm. These morphological traits correspond to Colletotrichum higginsianum. For pathogenicity tests, three 4-month-old L. draba plants were sprayed until runoff with a 106 conidia/ml suspension of the fungus and incubated for 72 h in plastic bags at 20 to 25°C in a quarantine greenhouse. Within 4 days, water-soaked lesions appeared that coalesced, resulting in chlorosis and collapse of inoculated leaves. Such symptoms are typical of infection by C. higginsianum and similar necrotrophic species (4). Fungi isolated from inoculated leaves were identified as C. higginsianum. To assess the host range of C. higginsianum, three plants each of turnip, radish, mustard greens, kale, broccoli raab, and Chinese cabbage, all in the Brassicaceae to which L. draba belongs, were inoculated with the same conditions used for the pathogenicity tests. Control plants in pathogenicity and host range tests were sprayed with sterile distilled water and all experiments were repeated at least once. All control plants were symptomless. Leaf necrosis occurred on radish and turnip and to a lesser extent on the lower leaves of Chinese cabbage and broccoli; numerous scattered dark necrotic flecks and small grayish leaf spots occurred on kale and mustard greens, respectively. These results are similar to previous studies (2,3) involving a cultivated species as the host in the field. The ITS1, 5.8S, and ITS2 sequences of this fungus (GenBank No. HM044877) were 99% similar to sequences of multiple isolates of C. higginsianum (GenBank Nos. AB042302, AB042303, AB455253, AJ558109, and AJ558110). To our knowledge, this is the first report of C. higginsianum on a wild species of the Brassicaceae, although a Colletotrichum sp. was reported on wild radish in Australia (1). References: (1) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (2) R. OConnell et al. Mol. Plant-Microbe Interact. 17:272, 2004. (3) R. P. Scheffer. N. C. Agric. Exp. Stn. Tech. Bull. 1950. (4) H. Sun and J. Z. Zhang. Eur. J. Plant Pathol. 125:459, 2009.