Effects of solar treatment on germination of sclerotia (Sclerotium rolfsii Sacc.) and on other soil mycoflora

Summary Only 10% of sclerotia germinated in a tarpcovered plot after 45 days of treatment while 50% of sclerotia germinated in the control plot. However, complete inhibition of sclerotia germination was not achieved. The maximum temperature recorded was 53°C, after 45 days of treatment in a tarped plot at 1 cm depth. The incidence of Acrophialophora fusispora, Aspergillus spp., Spicaria sp., and Trichoderma viride increased in soil after 45 days of the solar treatment.     

Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil

The colonization of Macrophomina phaseolina sclerotia by microbial parasites was evaluated in unsterilized field soil at different levels of soil moisture (0,-5, and-10 kPa) and temperature (20, 30, and 40°C). The maximum colonization of sclerotia was recorded in soil held at-5 or-10 kPa at 30–40°C. Trichoderma harzianum isolate 25–92 and Pseudomonas fluorescens isolate 4–92 were recorded as potential sclerotial parasites, and they significantly (P=0.05) reduced the germination of sclerotia by 60–63%. Cells of P. fluorescens and buffer-washed conidia of T. harzianum were completely agglutinated at 28°C with crude agglutinin of M. phaseolina. The ability of different antagonists to parasitize the sclerotia were correlated with the agglutination ability of the antagonists.     

Potential of Trichoderma spp. as consistent plant growth stimulators

In a series of repeated trials, six Trichoderma spp. strains, applied as a dried powder from a liquid fermentation in molasses/yeast medium, proved to be consistent at promoting the growth of lettuce (Latuca sativa L.) seedlings grown in a peat-sand potting compost in the glasshouse. Strains WT, 92, 20, and 75 at 0.75% or 1% w:w concentrations increased shoot dry weight by up to 26%, although WT did inhibit germination. For example, after 4 days only 13% of seeds sown in WT 1% w:w treated compost had germinated, whereas in other treatments germination was consistently greater than 32%. WT increased shoot fresh and dry weights by 14.3 g and 0.6 g per pot, respectively, without affecting the root dry weights, to give concomitant increases in shoot: root ratios of fresh and dry weight. The potential use of these Trichoderma spp. strains for plant growth promotion is discussed.     

苹果树腐烂病拮抗细菌鉴定及其抑菌作用效果测定

为了开发一种高效低毒的苹果树腐烂病生防制剂,通过对峙培养法、形态学及分子生物学的方法进行了苹果树腐烂病菌拮抗菌的分离筛选及鉴定,采用离体枝条法测定了拮抗菌对苹果树腐烂病的防效,并采用显微观察和液体培养法分别研究了拮抗菌对苹果树腐烂病菌的抑菌机理和无菌滤液对苹果树腐烂病菌生长的影响。分离筛选结果表明,从甘肃省各苹果产区果园土壤和苹果树枝条上分离得到23株细菌,2株对苹果树腐烂病菌具有较好拮抗作用,分别为LZ-1201和TS-1203,其对苹果树腐烂病菌菌丝生长抑制率分别为79.00%和85.00%。鉴定结果表明,菌株LZ-1201和TS-1203分别为枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。离体枝条防效测定表明,拮抗菌无菌滤液对苹果树腐烂病的防效随着稀释倍数增大而降低,原液防效最高,分别为74.43%和77.07%。抑菌作用机理结果表明,两株拮抗菌均可导致苹果树腐烂病菌丝膨大畸形、原生质浓缩、释放及溶解。拮抗菌无菌滤液对腐烂病菌生长的影响测定结果表明,无菌滤液对腐烂病菌分生孢子萌发和菌丝生长量均有显著抑制作用(P0.05),其无菌滤液稀释40倍时对腐烂病菌分生孢子萌发和菌丝生长量的抑制率均高于60%,表明该拮抗菌具有很好的生防潜力。     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

生物质炭介导生防微生物抑制辣椒疫霉的作用

生物质炭可有效防控土传病害,筛选并鉴定出生物质炭介导下的生防微生物,可为研究生物质炭防病机理和强化生物质炭防病效果提供理论依据。本研究首先进行秸秆生物质炭防控辣椒疫病盆栽试验,利用定量PCR和平板计数明确生物质炭在防控辣椒疫病时可富集的已知生防微生物,再通过选择性培养基初筛和定殖复筛筛选出生物质炭可富集的潜在生防微生物菌株,最后研究各菌株在土壤中对辣椒疫霉的抑制作用。结果表明,秸秆生物质炭使根际辣椒疫霉数量显著降低95.1%、辣椒疫病发生率显著降低91.1%,并使具有生防功能的木霉菌、青霉菌、曲霉菌、芽孢杆菌、假单胞菌和鞘氨醇单胞菌数量显著增加2.22倍、4.09倍、3.89倍、2.45倍、1.45倍和1.30倍。通过平板初筛得到可能被生物质炭富集的22株潜在生防菌株。定殖复筛剔除部分假性生物质炭介导菌株,获得可明确被生物质炭富集的2株木霉菌、3株青霉菌、2株曲霉菌、3株芽孢杆菌、3株假单胞菌、3株链霉菌和2株鞘氨醇单胞菌。木霉菌（TR1和TR3）、青霉菌（PE1）、曲霉菌（AS1和AS2）、芽孢杆菌（BA1、BA2和BA3）、假单胞菌（PS1和PS3）、链霉菌（ST1、ST4和ST5）13个菌株可显著削减土壤辣椒疫霉数量。其中,所有木霉菌和曲霉菌菌株（TR1、TR3、AS1和AS2）及芽孢杆菌（BA1和BA2）、假单胞菌（PS1和PS3）和链霉菌（ST1）9个菌株与生物质炭具有显著的协同抑制辣椒疫霉效果。因此,防控辣椒疫病时,木霉菌、曲霉菌、芽孢杆菌、假单胞菌和链霉菌是生物质炭介导下的主要防病微生物。     

Nitrogen-fixing potential of Acacia mangium and Acacia auriculiformis seedlings inoculated with Bradyrhizobium and Rhizobium spp.

Summary Two Australian Acacia species, A. mangium and A. auriculiformis were inoculated in vitro with eight strains of Bradyrhizobium spp. and two strains of Rhizobium spp. On the two plant species, only Bradyrhizobium spp. strains formed effective N₂-fixing nodules. A. mangium, which nodulates effectively with a restricted range of Bradyrhizobium spp. strains, is a specific host compared to A. auriculiformis. A. auriculiformis is assumed to be a promiscuous host because it nodulates effectively with a wide range of Bradyrhizobium spp. strains. Nodule efficiency as expressed by the ratio of N₂ fixed to nodule dry weight appeared to be higher in A. auriculiformis (0.44–0.81) than in A. mangium (0.23–0.55).     

Nodule-specific expression of Rhizobium meliloti symbiotic promoters P1 and P2 in chickpea-Rhizobium sp. symbiosis

Developmentally specific expression of Rhizobium spp. genes involved in symbiotic N₂ fixation is known to operate through cascade regulation of various nif and fix operons. Fusion constructs of lacZ under symbiotic promoters P1 (for nifHDK operon) and P2 (for fixABCX operon) of Rhizobium meliloti were mobilized into Rhizobium spp. (Cicer) strains Rcd301 and RCR13. The assays for -galactosidase activity to monitor the expression of lacZ under these promoters was performed in host backgrounds of Escherichia coli, R. meliloti, and Rhizobium spp. (Cicer). The enzyme assays indicated significant levels of expression from P1 and P2 promoters in chickpea rhizobia, specifically in symbiotic cells from nodules. However, as in R. meliloti, these promoters did not induce strong expression in free-living cells of Rhizobium spp. (Cicer). This indicates functional homology of R. meliloti promoters in rhizobium spp. (Cicer). Functional cross-reactivity of trans regulatory factors like NtrA, NtrC, and NifA between these rhizobia seems evident from the nodule-specific expression of P1 and P2 cis elements.     

利用微生物防除根寄生杂草列当

根寄生杂草列当(Orobanche spp.)已经严重制约全球许多地区的农业发展,寻找有效防除措施迫在眉睫。由于列当具有特殊生活史且与寄主关系密切,常规防除杂草措施难以达到理想防效。目前,尚无既能有效防除列当又不对寄主造成危害且便于大规模推广应用的列当防除措施。在众多列当防除措施中,微生物防除越来越引起关注和重视。本文对微生物防除列当的国内外研究进展及防除机理进行了综述。目前,列当生防微生物的研究主要集中在镰刀菌(Fusarium spp.)等列当病原菌和根瘤菌(Rhizobium spp.)等列当寄主植物共生菌上。微生物防除列当的机制主要包括两方面:一是通过产生代谢产物直接影响列当的萌发和生长,或通过降解列当种子萌发诱导物质间接影响列当的萌发;二是通过提高寄主植物自身对列当的抗性间接影响列当的寄生和生长。此外,本文还重点介绍了植物土传病害的土壤拮抗微生物防除列当杂草的可行性及研究进展。植物土传病害病原菌和列当均首先通过在地下侵染作物的根系进而危害作物正常生长,而作物抗土传病害的机理也与抗列当的机理类似。因此,存在于土壤中具有防治植物土传病害能力的微生物可能也具有防除根寄生杂草列当的功能。本团队前期试验从植物土传病害的土壤拮抗微生物中筛选到在盆栽试验中能够有效防除向日葵列当(O.cumana Wallr.)和瓜列当(O.aegyptiaca Pers.)的放线菌各1株,分别为淡紫褐链霉菌(Streptomyces enissocaesilis Sveshnikova)和密旋链霉菌(Streptomyces pactum Bhuyan B.K)。其中,密旋链霉菌的菌剂在田间试验中既降低了瓜列当的出土数量又增加了番茄的产量。总之,微生物是防除根寄生杂草列当的一条有效途径。     

Aluminum concentrations in sclerotia from a buried humic horizon of volcanic ash soils in Mt. Myoko,Central Japan

The morphology and chemical composition of fungal sclerotium-like grains collected from the buried humic horizon of volcanic ash soil in Mt. Myoko were examined using a scanning electron microscope (SEM), energy dispersion xray micro-analyzer (EDX), and electron probe micro-analyzer (EPMA). A high C content, low level of Si and high concentration of Al characterized the grains considered to correspond to the sclerotia of Cenoccocum graniforme. The ²⁷Al MAS NMR spectrum of the sclerotia showed the existence of 6- and 4-coordinated Al. X-ray diffraction analyses supported the presence of amorphous Al (ex. Al-humus complex) in the sclerotia.     

Differential responses of chickpea (Cicer arietinum L.) — Rhizobium combinations to saline soil conditions

Summary Chickpea cultivars (Cicer arietinum L.) and their symbiosis with specific strains of Rhizobium spp. were examined under salt stress. The growth of rhizobia declined with NaCl concentrations increasing from 0.01 to 2% (w : v). Two Rhizobium spp. strains (F-75 and KG 31) tolerated 1.5% NaCl. Of the 10 chickpea cultivars examined, only three (Pusa 312, Pusa 212, and Pusa 240) germinated at 1.5% NaCl. The chickpea — Rhizobium spp. symbiosis was examined in the field, with soil varying in salinity from electrical conductivity (EC) 4.5 to EC 5.2 dSm^-1, to identify combinations giving satisfactory yields. Significant interactions between strains and cultivars caused differential yields of nodules, dry matter, and grain. Four chickpea — Rhizobium spp. combinations, Pusa 240 and F-75 (660 kg ha^-1), Pusa 240 and IC 76 (440 kg ha^-1), Pusa 240 and KG 31 (390 kg ha^-1), and Pusa 312 and KG 31 (380 kg ha^-1), produced significantly higher grain yields in saline soil.     

Interactions between predatory ground beetles, the winter moth and an introduced parasitoid on the Lower Mainland of British Columbia

This study describes assemblages of predatory ground beetles in native birch forest and derived blueberry farms on the Lower Mainland of British Columbia and investigates their role in the successful biological control of winter moth (Operopthera brumata L.) by the parasitoid Cyzenis albicans Fallén. Pitfall traps at four sites in 1991 and 1992 indicated that natural birch forest was dominated by native carabid species, while open blueberry was dominated by introduced species and some native xerophilous species. Predation by generalist invertebrates was an important mortality factor for both winter moth and C. albicans at all sites and was greatest in the first 2 weeks after pupation. Ground beetles including Harpalus affinus (Schrank) and Pterostichus spp. consumed winter moth pupae in arenas but adult beetles rarely consumed C. albicans pupae. Much of the overall predation was due to smaller invertebrates including beetle larvae. Predators actively searched for buried pupae and selected among pupal types based on condition (dead, parasitised or healthy) and size. Prey-selection differed among sites but C. albicans pupae were preferred at three of the four sites. At the remaining site, an organic blueberry farm, over 90% of both parasitised and healthy pupae were consumed. High levels of predation did not affect the levels of parasitism by C. albicans at the sites. This herbivore–parasitoid–predator system demonstrates the importance of naturally occurring predatory invertebrates for successful integrated pest management, even where they prey upon a biological control agent.     

Symbiotic effectiveness of streptomycin-resistant Rhizobium spp. mutants of chickpea (Cicer arietinum L.)

Summary Antibiotic-resistant Rhizobium spp. strains have been used in ecological studies of legumerhizobia symbiosis. It has been suggested that in the course of acquiring resistance against high doses of antibiotics, rhizobia might lose their symbiotic effectiveness. Evidence both for and against this argument has been presented (Kremer and Peterson 1982; Materon and Hagedron 1983). This communication reports our experience with streptomycin-resistant (Str⁺) mutants of chickpea Rhizobium spp. strains. Parent strains were used as controls.Research paper No. 5233 from the Experiment Station, G.B.P.U.A.&T. Pantnagar, Nainital     

Chitinolytic and proteolytic activity of streptomycetes isolated from root-free soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary We tested 75 strains of Streptomyces spp. (25 taken from each environment of soil, rhizosphere, and mycorrhizosphere of pine, Pinus sylvestris L.) and all exhibited chitinolytic activity and hydrolysed gelatine and sodium caseinate in agar media. Enrichment of these media with glucose and NH₄NO₃ caused induction or stimulation of proteolytic Streptomyces spp. strains (80%) derived from root-free soil; inhibition of this activity was observed in most strains (92%) isolated from the root zone. The post-culture liquids of the rhizosphere strains cultured in the absence of glucose revealed a significantly higher proteolytic activity than those obtained from the root-free soil. The addition of glucose to the medium stimulated proteolytic activity in the post-culture broth of Streptomyces strains derived from soil and the mycorrhizosphere.     

Evaluation of nitrogen fixation by different strains of theAzolla-Anabaena symbiosis in the presence of a high level of ammonium

Summary The proportion of N derived from N₂ fixation for 99 strains ofAzolla spp. (comprising all known species) in the presence of ammonium (40 mg/1) was assessed using a¹⁵N-dilution technique. The percentage of N derived from air varied from 29.5% to 79.9%. Although the N concentration ofAzolla spp. was not correlated with fertilizer N, it correlated fairly well with N₂ fixation. Regression analysis suggests that the N yield ofAzolla spp. is more dependent on N₂ fixation than on ammonium assimilation. The high correlation between N yield and isotopically determined, fixed N₂ indicates that the N yield could be used as a parameter in the selection ofAzolla spp. strains that are capable of maintaining high N₂ fixation in the presence of a high level of ammonium.     

Distribution of Aspergillus section Flavi in soils of maize fields in three agroecological zones of Nigeria

Fungal communities in soils of Nigerian maize fields were examined to determine distributions of aflatoxin-producing fungi and to identify endemic atoxigenic strains of potential value as biological control agents for limiting aflatoxin contamination in West African crops. Over 1000 isolates belonging to Aspergillus section Flavi were collected from soil of 55 Nigerian maize fields located in three agroecological zones by dilution plating onto modified Rose Bengal agar. The most common member of Aspergillus section Flavi (85% of isolates) was the A. flavus L-strain followed by the unnamed taxon known as strain S_BG (8%), A. tamarii (6%) and A. parasiticus (1%). Highest incidence of S_BG was in Zaria district, and lowest was in Ogbomosho and Ado-Ekiti districts. Only 44% of 492 A. flavus isolates produced aflatoxins in liquid fermentation (limit of detection 5 ng g⁻¹). Thirty-two percent of the A. flavus isolates produced >1 μg g⁻¹ total aflatoxins but no A. flavus isolate produced G aflatoxins. When the agroecological zones were compared, significantly (P < 0.05) greater proportions of aflatoxigenic A. flavus isolates were found in the Northern Guinea Savannah (61%) than in Southern Guinea Savannah (31%). The Derived Savannah was intermediate between the other two agroecological zones. Each of the regions had atoxigenic strains of potential value as biological control agents. All S_BG and A. parasiticus isolates produced both B and G aflatoxins and greater than 300 μg g⁻¹ total aflatoxins. S_BG and A. parasiticus isolates were the greatest contributors to the aflatoxin-producing potential of fungal communities in regions where these isolates occurred.     

Biological control of Rhizoctonia sp. root rot of Casuarina equisetifolia seedlings by Frankia spp. strains

Seventy Frankia spp. strains (nodulating N₂-fixing actinomycetes) were isolated from root nodules of Casuarina equisetifolia from different localities of Tamil Nadu state, India. From these, four strains (UMCe12, UMCe23, UMCe35, and UMce55) were selected. Their potential use as biological control agents for Rhizoctonia solani root rot disease of C. equisetifolia seedlings and their relative efficiency in nodule production were investigated. Between the two inoculum broadcast systems tested, seed-coating with Frankia spp. cell suspension was superior to the soil application of cells as sand-vermiculite-basal ammonium propionate inoculum. UMCe12 was the promising strain, offering the highest level of disease protection (81.1%) and nodule production (88.1%) in the R. solani-infested soil, followed by UMCe23 (60.3 and 65.5% of disease protection and nodule production, respectively), UMCe55 (53.5 and 58.2%), and UMCe35 (45.4 and 44.5%). Further, a significant positive correlation was observed between the dose of Frankia spp. and efficiency in both disease control and nodule production.     

Effect of high temperature on plasmid curing of Rhizobium spp. in relation to nodulation of pigeonpea [Cajanus cajan (L.) Millsp.]

Summary Fifty-six isolates of Rhizobium and Bradyrhizobium spp. (Cajanus) were studied for their plasmid profile and N₂-fixation efficacy. One to three plasmids were reproducibly detected in all the Rhizobium spp. strains but no plasmid was detected in the Bradyrhizobium spp. strains. Rhizobium sp. strain P-1 was mutagenized by Tn5 and three nod^– and six nod⁺fix^– were screened for symbiotic parameters. Neomycin-sensitive mutants were isolated by elevated temperatrue (40°C) from tranconjugants carrying Tn5 insertions. The high temperature cured these mutants from the single large plasmid present in the parent strain P-1. All these cured mutants were nod^–, indicating that the genes for nodulation were present on this plasmid, which is readily cured at a high temperature (40°C). The high temperature in the semi-arid zones of Haryana could be responsible for the low nodulation of pigeonpea because the plasmid carrying the nodulation genes is cured at 40°–45°C giving rise to non-nodulating mutants.     

Effects of chitin amendment of soil on microorganisms,nematodes, and growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.)

Effects of soil amendment with crabshell chitin on the growth of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.), and on populations of soil bacteria, fungi, and plant-parasitic and free-living nematodes were investigated in a pot trial. Five soil samples were collected from Te Puke (Paengaroa Shallow Sand, a Typic Hapludand) and five from Hamilton (Bruntwood silt loam, an Aquic Hapludand), New Zealand. Subsamples of each soil were either amended with chitin or unamended and planted with white clover and ryegrass. The ryegrass shoot weight in amended soil was greater (P<0.01), most probably due to N mineralised from chitin. A significantly lower (P<0.01) root: shoot ratio of ryegrass in the amended soil also suggested improved N availability, and therefore less root mass was needed to support a given shoot mass. A reduction in nodulation was observed in 12-day-old white clover seedlings (P<0.05) and also in 6-week-old seedlings (P<0.01). The shoot weight of white clover was significantly lower (P<0.05) in amended soil, possibly due to phytotoxic effects of chitin. Chitin increased (P<0.01) the populations of bacteria and fungi by 13-fold and 2.5-fold, respectively. The cyst nematode of white clover, Heterodera trifolii, was significantly reduced in chitin-amended soil, possibly due to increased levels of chitinase produced by rhizosphere microorganisms. Two other plant-parasitic nematodes, Pratylenchus spp. and Tylenchus spp., were also reduced in ryegrass roots and in soil as a result of the chitin amendment. However, the total number of free-living nematodes increased 5.4-fold in amended soil.     

Gram-negative bacterial flora on the root surface of wheat (Triticum aestivum) grown under different soil conditions

We identified 161 Gram-negative bacterial strains isolated from the root surface of wheat grown under different soil conditions. The strains were divided into seven groups based on major morphological and physiological properties. Taxonomic allocation of the groups was verified by guanine+cytosine contents of DNA. Except for one group, which may be assumed to include bacteria belonging to the genera Flavobacterium and Cytophaga, the various groups were taxonomically united. The distribution of the groups changed with soil improvement. Pseudomonads predominated in unimproved soil, but Flavobacterium and Cytophaga spp. were predominant in the most improved soil. As all the strains were non-fermentative by Hugh and Leifson's test, API 20NE identification was applied. However, many strains were misidentified by this system, especially in the Flavobacterium and Cytophaga spp. group. For ecological studies, the strains were classified to species level by the API 20 NE system and by the results of a combination of guanine+cytosine (mol%) and isoprenoid quinone data. The pattern of distribution of the bacteria on the root surface of wheat varied at species level within one genus depending on soil conditions.Dedicated to Professor J. C. G. Ottow on the occasion of his 60th birthday