Increasing floral diversity for selective enhancement of biological control agents: A double-edged sward?

Floral resource subsidies can have differential effects on insect herbivores compared with the herbivores’ natural enemies. While the nectar of many plant species enhances parasitoid fitness, it may also increase damage by herbivores. This may occur as a result of enhanced herbivore fitness or by enhancing fourth-trophic-level processes, possibly disrupting a trophic cascade as a result. The responses of different arthropod guilds to different floral resource subsidies were compared using Plutella xylostella (Hyponomeutidae), its parasitoid Diadegma semiclausum (Ichneumonidae) and data from two other published herbivore–parasitoid systems. These were Dolichogenidea tasmanica (Braconidae) and its host Epiphyas postvittana, and Copidosoma koehleri (Encyrtidae) and its host Phthorimaea operculella. The parasitoids and hosts in the three systems exhibited differential responses to the nectar sources. The differential response was not explained by morphology, demonstrating that physical access to nectaries alone does not determine the potential of flowers as a food source. For some flowering plants, enhancement of herbivore and parasitoid fitness occurred. Other flowering plants, such as buckwheat and phacelia, conferred a selective enhancement on parasitoids by increasing only their fitness. More effective conservation biocontrol may be achieved by the provision of selective floral resources. Attempts to ‘engineer’ agroecosystems to enhance biological control require an extensive knowledge of the ecology of the herbivore, its enemies and their interactions with potential resource subsidies.     

Efficacy of inoculative releases of Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) against European corn borer Ostrinia nubilalis (Lepidoptera: Crambidae) in field corn

We evaluated the egg parasitoid Trichogramma ostriniae (Hymenoptera: Trichogrammatidae) to control European corn borer [Lepidoptera: Crambidae: Ostrinia nubilalis (Hübner)] in field corn in 2001 and 2002. Inoculative releases of 75,000 T. ostriniae/ha occurred in New York and Virginia in 5–10 cornfields per state when corn was at mid-whorl. Incidence of egg mass parasitism, number of stalk tunnels, incidence of ear damage, and whole-plant yield were evaluated. Parasitism of European corn borer egg masses ranged from 0 to 75% in release plots and was greater in release plots than in control plots. Individual comparisons between paired release and control plots showed no reductions in either stalk or ear damage. However, when data were combined across both years and fields, stalk and ear damage were significantly reduced in New York. In Virginia, no significant differences were detected using data obtained from one year. There were no differences in yield between release and control plots. Low densities of European corn borer, drought conditions in 1 year, and a larger plant canopy in field corn are possible reasons why T. ostriniae releases provided less control than has been observed in previous trials in sweet corn. Additional research focused on improved timing and frequency and number of releases is warranted.     

Biology of Lilioceris spp. (Coleoptera: Chrysomelidae) and their parasitoids in Europe

The biology and parasitoid complex of the lily leaf beetle (LLB), Lilioceris lilii Scopoli, and two congeneric species were investigated in Europe, as part of a biological control program against the LLB in North America. Eggs, larvae, and adults of L. lilii were collected in several countries in Europe, on both cultivated and wild Lilium spp., and reared in the laboratory and under natural conditions. Parasitoids were obtained and their biologies were studied. Similar investigations were made in Switzerland on two closely related species Lilioceris tibialis (Villa) found on wild Lilium spp., and Lilioceris merdigera (L.) on several other Liliaceae. The three species are strictly univoltine. Adults overwinter and lay eggs on leaves in early spring. The three beetle species have four instars, which were characterized by their head capsule width. Pupation occurs in a cocoon in the soil. Adults emerge in late summer and start feeding before reaching overwintering sites. Egg and larval parasitoids were obtained. Eggs of L. lilii and L. merdigera were parasitized by the mymarid Anaphes sp., a multivoltine species that needs alternate hosts for overwintering. Larvae were heavily attacked by several parasitoids, among which the most abundant were three ichneumonids, Lemophagus pulcher (Szepligeti), L. errabundus (Gravenhorst), and Diaparsis jucunda (Holmgren), and the eulophid Tetrastichus setifer Thomson. All four parasitoid species were found in the three beetles and in most European regions, but strong variations were observed in their relative abundance among hosts and geographic regions. Three of the four main larval parasitoids are strictly univoltine, whereas L. pulcher has a partial second generation. Lemophagus spp. are frequently parasitized by the ichneumonid hyperparasitoid Mesochorus lilioceriphilus Schwenke. Further details of the biology of the parasitoids are described, and their potential as biological control agents is discussed.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Post hoc assessment of an operational biocontrol program: efficacy of the flea beetle Aphthona lacertosa Rosenhauer (Chrysomelidae: Coleoptera), an introduced biocontrol agent for leafy spurge

Mixed populations of Aphthona lacertosa and Aphthona czwalinae were released at more than 50 locations in Alberta in 1997. Two and 3 years post-release, beetle populations were primarily A. lacertosa, with A. czwalinae forming less than 0.5% of the sampled populations. Beetle densities were moderate (10–70 beetles per m²) or high (>70 beetles per m²) at 14% and more than 60% of the sampled sites in 1999 and 2000, respectively. Larger beetles had greater instantaneous egg loads (r²=0.424,P=0.003). In 2000, the largest beetles were found at moderate density sites and there was a significant negative relationship between beetle size and the time taken to accumulate a degree day threshold of 1230 (for females: r²=0.678,P=0.001). Sites with the most rapid accumulation of degree days have the greatest potential for beetle population growth based on potential fecundity. Changes in leafy spurge percent cover, stem density, and canopy height from 1997 to 2000 were assessed across sites with low (<10 beetles per m²), moderate, and high beetle densities in 2000. Sites with high beetle densities had significantly greater reductions of leafy spurge within 5 m of the release point than sites with low beetle densities (P<0.017). Damage caused by the beetles at high-density sites was often visible as a halo-shaped patch of dead leafy spurge stems. The significant overall reduction of leafy spurge within release patches makes A. lacertosa a promising biocontrol agent for leafy spurge in Alberta.     

Effects of fine-grain habitat complexity on egg parasitism by three species of Trichogramma

Effects of microhabitat complexity on host foraging by three species of Trichogramma (T. deion, T. ostriniae, and T. pretiosum) were evaluated under simulated stored product conditions. All three species have been considered as potential biological control agents for the Indianmeal moth, Plodia interpunctella, in retail stores and warehouses. Trials were conducted with single naïve female parasitoids in 10-cm Petri dishes that were either empty, contained flour, or contained millet. Empty 15-cm Petri dishes, which served as a surface area control, comprised a fourth treatment. Females were allowed to forage for sentinel egg disks for 2 h, after which percentages of parasitism and egg mortality were computed. In addition, behavioral observations were made on a subset of the trials. Trichogramma deion parasitized more eggs than the other two species in the empty dishes and in the dish containing flour. For T. deion, rates of parasitism and egg mortality were significantly greater in both the small and large empty dishes than in the small dishes containing flour or millet. Parasitism was consistently low for all three species in the grain-filled dishes. Among species, T. ostriniae spent the most time walking, while T. pretiosum was the most sedentary. In addition, T. pretiosum spent significantly more time on the first egg visited compared with subsequent eggs. T. deion may be the best-suited for use as a biological control agent for P. interpunctella. However, the potential negative effects of fine-grain habitat complexity should be taken into account when developing a release protocol for Trichogramma spp.     

Survey of brown soft scale Coccus hesperidum L. parasitoids in southern California citrus

The parasitoid complex of brown soft scale, Coccus hesperidum L., a multivoltine soft scale, was determined in southern California citrus over the period February 2004–March 2006. The survey was conducted by placing brown soft scale-infested yucca leaves in the canopy of citrus trees and subsequently rearing individually isolated parasitized scales in the laboratory. A total of 14 species parasitized brown soft scale in the field, the most abundant ones belonging to the genus Metaphycus Mercet (75%). The most abundant parasitoid species was Metaphycus angustifrons Compere (38% parasitism), and this is a new record of establishment for this species in California. Coccophagus species accounted for only 11% parasitism. There were important spatio-temporal differences across the parasitoid complex survey locations. We also found that the five most abundant encyrtid parasitoid species showed preferences for scales of different sizes. Our results have implications for biological control of citricola scale, Coccus pseudomagnoliarum (Kuwana), an important pest of citrus in the San Joaquin Valley of central California. Notably, this species is nearly absent in southern California. Brown soft scale is considered to be an alternate host for parasitoids of citricola scale, a univoltine soft scale, at times when the latter species is unavailable for parasitism.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Measuring parasitoid movement from floral resources in a vineyard

The movement of natural enemies from floral resources is of particular importance in habitat manipulation research, as the distances that they disperse have consequences for the deployment of floral resources to improve insect natural enemy fitness. A number of marking techniques can be used to measure natural enemy movement; however, many of these are labour-intensive and not appropriate for many natural enemy species, the alternative, self-marking techniques, are less common. The aim of this study was to determine whether rubidium chloride (RbCl) could be used to measure the movement of Dolichogenidea tasmanica (Cameron) (Hymenoptera: Braconidae) from flowering buckwheat, Fagopyrum esculentum Moench plants in an organic vineyard. D. tasmanica is the most common parasitoid of leafroller larvae, a serious pest of grapevines in Australia and New Zealand. Foliar applications of rubidium chloride were made to a single strip of buckwheat in the centre of each of five vineyard areas. Sticky traps were placed in each area at distances of 0, 4, 10 and 30 m in opposite directions from the buckwheat to collect adult D. tasmanica. D. tasmanica were marked with rubidium after buckwheat plants had been sprayed with RbCl and were trapped up to 30 m from the plants within a seven-day sampling period. This study indicates that RbCl can be used to mark parasitoids to measure their movement from floral resources and may be used to inform decisions on the deployment of appropriate flowering plant species in conservation biological control.     

不同品种番石榴花的挥发性成分分析

为探究番石榴(Psidium guajava)花挥发性成分组成,采用顶空/气相色谱-质谱联用技术对10个番石榴品种(‘翠玉’、‘帝王’、‘本土’、‘红叶’、‘粉红蜜’、‘珍珠’、‘西瓜’、‘水蜜’、‘木瓜’和‘红宝石’)花的挥发性成分进行鉴定分析。结果表明,10个品种共检出相对含量在0.1%以上的挥发性成分43种,包含共有成分10种,以萜烯类化合物(89.77%～97.40%)为主。β-石竹烯、β-罗勒烯、桉叶油醇和D-柠檬烯为花主要挥发性成分。影响品种间挥发性成分差异的成分主要有7种,分别为α-蒎烯、β-罗勒烯、D-柠檬烯、3-蒈烯、香树烯、β-长叶蒎烯和1-异丙基-4,7-二甲基-1,2,3,5,6,8a-六氢萘。按照香气相似性,‘翠玉’和‘帝王’归为一类,‘水蜜’和‘本土’归为一类,‘粉红蜜’、‘西瓜’、‘红叶’、‘木瓜’和‘红宝石’归为一类,‘珍珠’为单独一类。不同品种番石榴花挥发性成分存在相似性和差异性,为番石榴花混合采摘及后期个性化开发利用提供理论基础。     

Flower Abscission in Excised Inflorescences of Three Plectranthus Cultivars

Flower abscission induced by ethylene in three Plectranthus cultivars was investigated in order to characterise response to a range of inhibitory and antagonistic compounds. Excised inflorescences were exposed to 100 ml l⁻¹ ethylene gas or placed in various concentrations of ethephon (277, 27.7, 2.77, 0.277 and 0.0277 μM). Flower abscission in Plectranthus was readily induced by applying ethylene gas and by the 277 μM dose of ethephon. Removal of the inflorescences from the ethylene treatment prevented subsequent flower abscission. This implies that ethylene treatment did not induce an autocatalytic production of ethylene. Compounds that are known to compete for the ethylene receptor (100 and 500 ppb 1-methylcyclopropene or 100 and 500 ppm 2,5-norbornadiene) did not reduce abscission in this system. Also, application of the ethylene biosynthesis inhibitor, aminooxyacetic acid at 1 mM, was ineffective at preventing ethylene-induced flower abscission. In contrast, one compound known to block protein production (100 μM cycloheximide) and a non-competitive inhibitor of ethylene action (2 mM silver thiosulfate) did prevent ethylene-induced abscission. We conclude that flower abscission in cut inflorescences of Plectranthus is very likely mediated by endogenous ethylene production, but that control of ethylene-induced flower abscission in this genus can not be readily obtained by most ethylene antagonists that are known to be effective in other systems.     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

Muscodor albus, a potential biocontrol agent against plant-parasitic nematodes of economically important vegetable crops in Washington State, USA

The fungus, Muscodor albus, was tested for nematicidal and nematostatic potential against four plant-parasitic nematode species with three different feeding modes on economically important vegetable crops in the Pacific Northwest. Meloidogyne chitwoodi, Meloidogyne hapla, Paratrichodorus allius, and Pratylenchus penetrans were exposed for 72 h to volatiles generated by M. albus cultured on rye grain in sealed chambers at 24 °C in the laboratory. In addition, the nematodes were inoculated into soil fumigated with M. albus, and incubated for 7 days prior to the introduction of host plants under greenhouse conditions. The mean percent mortality of nematodes exposed to M. albus in the chamber was 82.7% for P. allius, 82.1% for P. penetrans, and 95% for M. chitwoodi; mortality in the nontreated controls was 5.8%, 7%, and 3.9%, respectively. Only 21.6% of M. hapla juveniles died in comparison to 8.9% in controls. However, 69.5% of the treated juveniles displayed reduced motility and lower response to physical stimulus by probing, in comparison to the control juveniles. This is evidence of nematostasis due to M. albus exposure. The greenhouse study showed that M. albus caused significant reduction to all nematode species in host roots and in rhizosphere soil. The percent mortality caused by M. albus applied at 0.5% and 1.0% w/w in comparison to the controls was as follows: 91% and 100% for P. allius in the soil; 100% for P. penetrans in bean roots and soil; 85% and 95% for M. chitwoodi in potato roots, and 56% and 100% in the soil; 100% for M. hapla both in pepper roots and soil. In this study, M. albus has shown both nematostatic and nematicidal properties.     

Studies ontrichogramma buesi as a biocontrol agent againstPieris rapae in Egypt

Trichogramma buesi was reared in the laboratory on eggs of the Mediterranean flour moth,Anagasta kuehniella. The incubation period of the parasite's egg was 27 h at 23 °C and 22 h at 27 °C. The larval stage lasted 3.6 and 3.2 days, the pre-pupa lasted 16 and 23 h, and the pupa lasted 5.4 and 4.6 days at 23° and 27°C, respectively. The total developmental time (from egg to adult) averaged 9.2, 9.4, and 9.1 days when the parasite was reared on eggs ofPieris rapae, Spodoptera littoralis, andA. kuehniella, respectively, at 27 °C. Sex ratio inT. buesi was 1 ♂: 1 ♀ in nature and 1 ♂: 1.3 ♀ in the laboratory. The daily and total numbers or progeny produced/female were 5.1 and 98.2 adults, respectively. The parasite female, fed on honey, lived 10.7 days at 27 °C and 12.1 days at 23 °C. Percentages of parasitism byT. buesi on eggs ofP. rapae collected from cabbage fields ranged between 0 and 31.5 % in 1985 and betwcen 0 and 36.4% in 1986 during July through December. The respective figures on eggs collected from turnip fields were 16–42.2% and 12.5–32.1% during November and December.      

Comparative analysis of the reproductive biology of two Peristenus species (Hymenoptera: Braconidae), biological control agents of Lygus plant bugs (Hemiptera: Miridae)

Peristenus digoneutis Loan and Peristenus stygicus Loan, parasitoids of the European tarnished plant bug Lygus rugulipennis Poppius, are established in the United States for biological control of native North American Lygus species, and are being considered for deliberate release in Canada. High lifetime fecundity of parasitoids is considered a desirable attribute of biological control agents and therefore, an understanding of parasitoid reproductive biology is required. In the present study, the potential lifetime fecundity of both agents was compared under laboratory conditions to estimate the potential impact of Peristenus species on Lygus. Synovigenic P. digoneutis and P. stygicus females oviposited most actively in the first two weeks of their lifetime, with a maximum average daily oviposition rate after five days. The maximum number of eggs laid per day was 83 eggs for P. stygicus, and 36 eggs for P. digoneutis. P. digoneutis has an average potential lifetime fecundity of 385 ± 35 SE eggs produced over 22 ± 3 SE days. In contrast, P. stygicus females have a 50% higher mean potential lifetime fecundity reaching 782 ± 65 SE eggs over 28 ± 1 SE days. A positive correlation between lifetime fecundity and body size was found only for P. stygicus, and both species showed a significant relationship between lifetime fecundity and oviposition period. The present study demonstrates that the fecundity of P. digoneutis and P. stygicus is considerably higher than previously reported. Based on these findings, P. stygicus appears to be the most effective biological control agent for Lygus lineolaris (Palisot de Beauvois) when only fecundity is taken into consideration.     

The potential for indirect effects between a weed, one of its biocontrol agents and native herbivores: A food web approach

The safety of biological control is a contentious issue. We suggest that constructing and analyzing food webs may be a valuable addition to standard biological control research techniques, as they offer a means of assessing the post-release safety of control agents. Using preliminary data to demonstrate the value of food webs in biocontrol programs, we quantified the extent to which a key agent has infiltrated natural communities in Australia and, potentially, impacted on non-target species. Using these data, we also demonstrate how food webs can be used to generate testable hypotheses regarding indirect interactions between introduced agents and non-target species. We developed food webs in communities invaded to varying degrees by an exotic weed, bitou bush, Chrysanthemoides monilifera ssp. rotundata, and a key biocontrol agent for this weed in Australia, the tephritid fly, Mesoclanis polana. Three food webs were constructed during springtime showing the interactions between plants, seed-feeding insects and their parasitoids. One food web was constructed in a plot of native Australian vegetation that was free of bitou bush (‘bitou-free’), another in a plot of Australian vegetation surrounded by an invasion of bitou bush (‘bitou-threatened’) and a third from a plot infested with a monoculture of bitou bush (‘bitou-infested’). The bitou-free web contained 36 species, the bitou-threatened plot 9 species and the bitou-infested web contained 6 species. One native Australian herbivore attacked the seeds of bitou bush. M. polana, a seed-feeding fly, was heavily attacked by native parasitoids, these being more abundant than the parasitoids feeding on the native seed feeders. A surprising result is that none of the three species of native parasitoids reared from M. polana were reared from any of the native herbivores. The food webs revealed how a highly host-specific biocontrol agent, such as M. polana has the potential to change community structure by increasing the abundance of native parasitoids. The webs also suggest that indirect interactions between M. polana and native non-target species are possible, these been mediated by shared parasitoids. The experiments necessary to determine the presence of these interactions are outlined.     

Trapping and sex pheromone-mediated flight and landing behaviour of maleCtenopseustis obliquana

A sex pheromone-baited delta trap was found to be inefficient at eliciting landing and entering of the trap by maleCtenopseustis obliquana. The inefficiency of the delta trap related to turbulence altering the pheromone plume and the concomitant effect on the flight manoeuvres of male moths. In the wind tunnel, high proportions of males flew upwind and landed on the sides, outside, of the trap, but only a relatively small proportion of these males entered the trap and contacted the sticky surface. When males approached the delta trap, they tended to fly in wide zigzags (i.e., large inter-track reversal distances) and at an altitude near the top of the trap, where the trap was relatively narrow in width (compared to the bottom). Thus, these flight manoeuvres largely precluded males from entering the trap. Greater numbers of male moths entered the trap when: (i) the front barriers of the delta trap were removed, (ii) pheromone dosage was increased to 300 μg, and (iii) the trap design was changed to a rectangular one. The first two changes appeared to influence the flight manoeuvres of males (who appeared to fly with narrower inter-track reversal distances), while the third change apparently did not affect the glight manoeuvres of males, but rather allowed more males to enter the trap because of the greater area of the entrance. The low trap catches of maleC. obliquana in the field are also a consequence of the glue which is currently used. After contact with this glue most males are able to escape, flying off the sticky surface and losing contact with the pheromone. A field trial found that delta traps with another glue caught more than three times the number of males of the related tortricid moth,Epiphyas postvittana, than delta traps with the currently used glue.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

Effectiveness of honey bees in delivering the biocontrol agent Bacillus subtilis to blueberry flowers to suppress mummy berry disease

Honey bees are important pollinators of commercial blueberries in the southeastern United States, and blueberry producers often use supplemental bees to achieve adequate fruit set. However, honey bees also vector the plant pathogenic fungus Monilinia vaccinii-corymbosi which infects open blueberry flowers through the gynoecial pathway causing mummy berry disease. Here, we report the results of a 3-year field study to test the hypothesis that using bee hives equipped with dispensers containing the biocontrol product Serenade, a commercial formulation of the bacterium Bacillus subtilis which has shown activity against flower infection by M. vaccinii-corymbosi in laboratory experiments, can reduce mummy berry disease incidence when honey bees are used as pollinators in blueberries. Individual honey bees carried 5.1–6.4 × 10⁵ colony-forming units (CFU) of B. subtilis when exiting hive-mounted dispensers with Serenade. On caged rabbiteye blueberry bushes in the field, population densities of B. subtilis vectored by honey bees reached a carrying capacity of <10³ CFU per flower stigma within 2 days of exposure, and there was a highly significant non-linear relationship between B. subtilis populations per stigma and bee activity, expressed as number of legitimate flower visits per time interval per cage (R = 0.6928, P < 0.0001, n = 32). Honey bee density (1600 or 6400 individuals per 5.8-m³ cage) and Serenade treatment (presence or absence of the product in hive-mounted dispensers) significantly (P < 0.05) affected the incidence of fruit mummification on caged bushes, whereby increasing bee density increased disease incidence and application of Serenade reduced disease levels. Taken together, results of this study suggest that use of a hive-dispersed biocontrol product such as Serenade as a supplement during pollination can reduce the risk of mummy berry disease. This may be a prudent practice that optimizes the benefits to pollination of high bee densities while reducing the associated disease-vectoring risk.