Plant colonization and environmental fate of the biocontrol fungus Phoma macrostoma

Several isolates of the fungus Phoma macrostoma demonstrated bioherbicidal activity against dandelion seedlings when applied to soil. Weed control ranged from 36 to 100% depending on the isolates and the doses applied. Using microbiological and molecular genetic techniques, the ability of these isolates to colonize target, and nontarget plants and to disperse and persist in soil were determined. PCR primers highly specific to the biocontrol isolates of P. macrostoma, were used to detect the isolates at rates of application between 4 and 1000 g/m². Based on the results from representative isolates tested, it was concluded that P. macrostoma colonized root tissues of both resistant and susceptible crop species and a susceptible weed species grown in treated soil, and the frequency of fungal isolation declined with time. It was occasionally detected on untreated plant tissues, which may have resulted from either natural occurrences on seed, or contamination of soil. The biocontrol fungus appeared to have limited mobility in the soil since it was not often detected away from the area where it was placed. It persisted in the soil at detectable levels for up to 4 months, but then its presence declined with time. One year post application, P. macrostoma was either not present or significantly reduced in both soil and plant samples depending on the year of sampling. The results suggested that the isolates of P. macrostoma used for biological weed control would have minimal environmental impact due to its ubiquitous nature, limited mobility, and weak persistence over seasons.     

Field evaluation of Tilapia zilli (Gervais) as a biological control agent for mosquito control

Tilapia zilli (Gervais), a larvivorous fish identified in the Mwea Rice Irrigation System (Kenya), was evaluated in the field to assess its potential for biological control of mosquito larvae. The study was conducted from October 1991 to April 1992 in experimental ponds constructed at Mbui-Njeru, which is one of the 36 villages in the irrigation scheme. T. zilli was found to be extremely effective in controlling mosquito breeding that persisted throughout the study period. However, the presence of vegetation and turbidity significantly reduced its efficiency in predation. The negative effects of vegetation could be averted by combining T. zilli with an herbivorous fish to control weeds. Similarly, breeding of other larvivorous organisms, including frogs, should be encouraged to enhance mosquito control efficiency. It is suggested that positive measures should be taken to deter colonization of catfish whose presence aggravates the turbidity problem and also depletes the fish population due to its substrate feeding and carnivorous behaviour.     

Assessment of Boeremia exigua var. rhapontica,as a biological control agent of Russian knapweed (Rhaponticum repens)

Russian knapweed (Rhaponticum repens (L.) Hidalgo) is an herbaceous perennial weed that was introduced and has become invasive in the United States, particularly in the semi-arid west. It is characterized by its extensive root system, low seed production, and persistence. The weed has caused serious reductions in yields and crop value and may significantly devalue the land itself. Conventional control strategies have been inadequate because of the size of infestations and economic and environmental costs of control. Biological control has been a sought-after potential solution to this weed problem. In the summer of 2002, diseased R. repens plants were collected near Cankiri, Turkey, and the facultative saprophytic fungus Boeremia exigua isolate FDWSRU 02-059 was isolated from diseased plants. Bayesian analysis of the actin, beta-tubulin, calmodulin, elongation factor, and ITS genes, of 66 isolates, representing the ten species of Boeremia and the 11 varieties of B. exigua, including FDWSRU 02-059, showed that the isolate is a unique genetic entity and was named B. exigua var. rhapontica Berner, Woudenberg & Tunali, var. nov. MycoBank MB809363. Disease incidence and severity data from host-range determination tests conducted at 25 °C, the optimum temperature for growth and sporulation of B. ex. rhapontica, with adequate dew periods, were combined with a genetic distance matrix based on ITS sequences of 66 plant species related to R. repens. The combined disease and genetic data were analyzed by mixed model equations to produce best linear unbiased predictors (BLUPs), standard errors, and P > |t| values, in t-tests against zero, for disease incidence and severity for each species. BLUPs of disease incidence were significantly different from zero only for three Rhaponticum spp. while BLUPs of disease severity rankings were significantly different from zero only for R. repens, Rhaponticum carthamoides, Rhaponticum uniflorum, and Leuzea berardioides. Best linear unbiased predictors for differences in above-ground dry weights between control and inoculated plants of a subset of the species evaluated were not significant. However, above-ground damage by B. ex. rhapontica to R. repens was nearly twice that for any other species, except Rhaponticum species.     

Discovery of a CI-inducing Wolbachia and its associated fitness costs in the biological control agent Aphytis melinus DeBach (Hymenoptera: Aphelinidae)

Wolbachia is an endosymbiotic bacterium that infects a large percentage of arthropods and can affect the fitness of its host. Here we verified for the first time that the biological control agent Aphytis melinus DeBach is infected with a Wolbachia that causes complete cytoplasmic incompatibility, and conducted an insectary and field survey to determine the infection frequency. A. melinus appears to suffer fitness costs associated with infection based on measurements of longevity and fecundity. We also quantified the Wolbachia titers of A. melinus reared at different temperatures and found that, although not completely cured, increased temperature resulted in a significant reduction in the number of Wolbachia copies found in an individual wasp. Implications of our results for biological control are discussed.     

Biology of Platycephala planifrons (Diptera: Chloropidae) and its potential effectiveness as biological control agent for invasive Phragmites australis in North America

Phragmites australis is a cosmopolitan clonal grass valued for its support of diversity-rich communities in its native range and feared for its devastating effects on native diversity where the species is introduced. Lack of successful control in North America resulted in the initiation of a biological control program. We used a combination of field surveys and common garden experiments in Europe to study life history and ecology of a chloropid fly, Platycephala planifrons, to assess its potential as a biological control agent. The fly is widely distributed (in non-flooded sites) throughout Eurasia but attack rates are generally low (mean 5–10%; max. 29%). Adults emerge in late June and may live for several months. Females lay eggs at the base of Ph. australis shoots. First instar larvae of this stem-feeding fly overwinter in dormant below-ground shoots of Ph. australis and rapidly complete development in early spring. Larval feeding destroys the growing meristem of the shoot causing premature wilting and 60–70% reductions in shoot biomass production. Early season attack and considerable impact suggest that Pl. planifrons could be a potent biocontrol agent, if it can escape suppression by natural enemies in the introduced range. However, the generally low attack rates in its native range and its dependence on dry sites appear to make the species a “second-choice” candidate for potential release in North America.     

Streptomyces neyagawaensis as a control for the hazardous biomass of Microcystis aeruginosa (Cyanobacteria) in eutrophic freshwaters

An aquatic bacterium capable of eliminating the cyanobacterium Microcystis aeruginosa was isolated from the sediment of an eutrophic lake (Lake Juam, Korea). On the basis of 16S rDNA sequences and biochemical and morphological characteristics, the isolate was determined to be Streptomyces neyagawaensis. It grew optimally at 40 °C and pH 7. In the presence of this bacterium, the biomass of cyanobacterium M. aeruginosa NIES-298 was strongly suppressed, by up to 84.5% in abundance compared to the control. The antialgal activity of S. neyagawaensis depended on the growth phase of the cyanobacterium, but not of the antialgal bacterium. The antialgal activity of S. neyagawaensis was effective against a wide range of algae, including the green alga Chlorella sp., the diatoms Aulacoseira granulata and Stephanodiscus hantzschii, and four cyanobacteria, M. aeruginosa NIES-44, Anabaena cylindrica, Anabaena flos-aquae, and Oscillatoria sancta. S. neyagawaensis indirectly attacked M. aeruginosa by secretion of extracellular antialgal substances that were localized in the bacterial periplasm and had a specific activity of 7.7 U/μg. These results suggest that indigenous bacteria isolated from sediments may have potential application in controlling harmful cyanobacterial blooms in freshwaters.     

Pre-release impact assessment of two stem-boring weevils proposed as biological control agents for Alliaria petiolata

Experimental studies can be useful tools to test plant responses to herbivory and to quantify the impact of potential biological control agents prior to their release. We evaluated the per-capita effect of Ceutorhynchus alliariae and C. roberti, two stem-boring weevils currently being investigated as potential biological control agents for garlic mustard, Alliaria petiolata, in North America. Weevils were released at three different densities in individual and mixed-species treatments onto potted plants of A. petiolata. Damage by C. roberti alone and by both weevils combined caused an increase in the numbers of inflorescences produced per plant. Although plants could compensate for low levels of damage, moderate to high levels of damage by both C. alliariae and C. roberti, individually and in combination, caused a decrease in plant height and a reduction in seed output per plant. The damage inflicted by both weevil species is similar so the overall impact of both species combined can be predicted by summing the impact of each species alone. Provided they are sufficiently host specific, both weevils could be released as biocontrol agents. Because reduced seed production is necessary to suppress A. petiolata populations, both species have the potential to contribute to control of A. petiolata in North America.     

The effect of manual defoliation and Macaria pallidata (Geometridae) herbivory on Mimosa pigra: Implications for biological control

Trials were conducted to test the effects of artificial defoliation and defoliation by Macaria pallidata (Warren) (Geometridae) larvae on the invasive weed Mimosa pigra L. Herbivory is generally thought to be detrimental to plant fitness but it is well documented that many plants can increase growth rates or reproduction to compensate for damage. The compensatory ability of an invasive plant has implications for the potential success of defoliating biocontrol agents. Mimosa compensated for 25% manual defoliation, but at 50% and 100% defoliation levels plants suffered a significant reduction in growth rate, height, stem diameter, and biomass. Defoliation by one cohort of macaria larvae, at densities of eight larvae per plant, significantly reduced growth rates and plant height after 1 week. There were no differences between the effects of macaria larvae and manually simulated defoliation. These results suggest that defoliating biocontrol agents can have a valuable role in mimosa control programs.     

Sphaceloma poinsettiae as a potential biological control agent for wild poinsettia (Euphorbia heterophylla)

Sphaceloma poinsettiae, a scab-causing fungus that attacks wild poinsettia (Euphorbia heterophylla) was evaluated as a mycoherbicide. Plants representing nine different E. heterophylla accessions were tested and variable resistance levels to 10 different isolates of S. poinsettiae were observed. One isolate was selected based on its high aggressiveness; it caused scab that led to stem-girdling in the majority of plants belonging to most wild poinsettia accessions. Plants inoculated with conidial suspension developed low disease severity; suspensions of mycelial fragments caused higher disease severity. Viability of mycelium declined rapidly either in storage under room temperature or at 4 °C. After 25 days, 60% of mycelial fragments kept in distilled water at 4 °C or in 35% sterile sucrose solution germinated. Cultures of S. poinsettiae were capable of growing at temperatures ranging from 10 to 30 °C (maximum growth at 25 °C) whereas the mycelial fragments germinated at 20–30 °C. The highest mycelial yield occurred in potato-dextrose broth after culturing for 12 days at 168 rpm at room temperature. A host-range test involving 37 plant species in addition to wild poinsettia indicated that S. poinsettiae is highly specific to E. heterophylla. The viability of S. poinsettiae as a mycoherbicide depends on the development of suitable mass production, formulation, and inoculum preservation methods. On several occasions, this fungus has been observed to cause devastating epidemics in the field on this host; therefore, it should be considered a potential biocontrol agent of wild poinsettia.     

Plant size preference of Agapeta zoegana L. (Lepidoptera: Tortricidae), a root-feeding biological control agent of spotted knapweed

Agapeta zoegana L. (Lepidoptera: Tortricidae) is an oligophagous herbivore that was introduced to North America as a biological control agent of spotted knapweed, Centaurea stoebe L. subsp. micranthos (Gugler) Hayek (often called Centaurea maculosa Lam.). Spotted knapweed is a perennial plant that usually increases in size each year. A previous field study reported that more larvae were found on larger plants and that infested plants tended to be larger than uninfested ones. Precisely quantifying the size-specific attack rate can help us model the impact of this agent on the weed population and better understand the interspecific interactions to improve the effectiveness of biological control. Field data were analyzed to determine the relative preference of attack for each size class of the host plant. Plants were classified based on root diameter at 2 cm below the root crown. Although small plants (<3 mm root diameter) were more abundant in the field population, the highest infestation rates occurred in large plants. Chesson’s electivity index was generally positive for root diameters >3.5 mm, indicating preferential attack of large plants. Because of its host-size preference, A. zoegana is expected to primarily affect large plants, which is contrary to previous expectations. Quantifying the insect’s direct impact is a difficult challenge, which may require several field seasons of measuring accumulated damage on individual plants. In order to complement the biological control agents already established, foreign exploration should focus on finding an agent that attacks young knapweed plants. These results also indicate that the efficiency of sampling roots in the field to detect the presence of A. zoegana can be improved by choosing only the largest plants.     

Prerelease efficacy assessment, in quarantine, of a tephritid gall fly being considered as a biological control agent for Cape-ivy (Delairea odorata)

Due to the long-standing emphasis on only releasing host-specific agents, classical biological control of weeds has an enviable track record of few direct impacts to nontargets. However, even an agent whose host-range is restricted solely to the target weed can have indirect impacts. Such indirect impacts are most likely if, after release, the populations of the agent build up to high numbers without causing accompanying declines in the populations of the target weed. Therefore, it is advisable, prior to release, to demonstrate that the candidate agent is not only host-specific, but that it has clear potential to depress populations of the target weed. Prerelease efficacy assessments (PREA) of potential weed biocontrol agents are not yet common, and are most easily done in the region where both the target and the potential agent are native. We present an example of a PREA performed under strict containment conditions of an approved quarantine facility. A gall-forming fly, Parafreutreta regalis, from South Africa is being considered for release in California to control Cape-ivy, Delairea odorata. We conducted two trials exposing test Cape-ivy plants to two different densities of this fly, and, after approximately two months, comparing the growth of the galled vines to similar vines that had not been exposed to flies. Under both the high density (10 pairs of flies/plant) and low density (2 pairs/plant) treatments, the galled vines exhibited visible stunting, and the ungalled stems were longer, and had more nodes and larger leaves. These trials confirmed that relatively subtle, sublethal impacts on the target can be quantified, even under strict containment conditions, and this should encourage others to assess, prior to release, the potential impact of prospective agents on their proposed target.     

生物制品原辅料及其质量控制

生物制品原辅料的质量控制是保证产品质量的重要因素,就生物制品原辅料分类及质量控制进行了阐述和归纳,并就国家对生物制品原辅料的质量控制要求进行了探讨。     

Classical biological control of Aphis gossypii (Homoptera: Aphididae) with Neozygites fresenii (Entomophthorales: Neozygitaceae) in California cotton

In August 1994 and 1995 classical biological control releases were made in cotton in the San Joaquin Valley, California, with an Arkansas strain of the entomopathogenic fungus, Neozygites fresenii, a pathogen of the cotton aphid, Aphis gossypii. Pre-release samples in both years indicated that N. fresenii was not naturally present in A. gossypii populations in the San Joaquin Valley. Two release methods were compared: dried N. fresenii-infected cotton aphid “cadavers” and chamber inoculation of A. gossypii. Both methods were successful in introducing N. fresenii to cotton aphids in California; however, higher prevalence of fungal infection resulted with the cadaver treatments. N. fresenii persisted and spread in the aphid population until early October 1994 and late September 1995. The highest mean percentage infection in the cadaver treatment in 1994 reached a level (14%) considered imminent for epizootics (12–15%). The use of predator exclusion cages resulted in higher N. fresenii prevalences.     

Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 10⁴ to 10⁸ conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05). With respect to fungal post-infection growth parameters, only germination and sporulation were significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.     

Costs of mass-producing the root weevil, Mogulones cruciger, a biological control agent for houndstongue (Cynoglossum officinale L.)

The cost of rearing the root-feeding weevil, Mogulones cruciger Herbst, to control the invasive weed houndstongue (Cynoglossum officinale L.) was determined for two managed production methods. Production in an insectary setting provides control over rearing and all adult weevils that emerge can be collected, but required facility investment and high labor input. Mass-rearing in a managed ‘field crop’ setting required less facilities and labor while the insects were multiplying, but capture of the emerged adults was challenging and labor intensive. Estimated per adult weevil production costs were $CDN 2.65 for the insectary approach, and from $CDN 0.10 to $CDN 0.14 for mass-rearing in the managed field crop setting. Even though collection of adult weevils in the field crop production system was challenging, commercial production of M. cruciger should consider use of this mass-rearing method because of its lower cost.     

The introduction and release of Chiasmia inconspicua and C. assimilis (Lepidoptera: Geometridae) for the biological control of Acacia nilotica in Australia

Two geometrid moths Chiasmia inconspicua and Chiasmia assimilis, identified as potential biological control agents for prickly acacia Acacia nilotica subsp. indica, were collected in Kenya and imported into quarantine facilities in Australia where laboratory cultures were established. Aspects of the biologies of both insects were studied and CLIMEX® models indicating the climatically favourable areas of Australia were developed. Host range tests were conducted using an approved test list of 74 plant species and no-choice tests of neonate larvae placed on both cut foliage and potted plants. C. inconspicua developed through to adult on prickly acacia and, in small numbers, Acacia pulchella. C. assimilis developed through to adult on prickly acacia and also in very small numbers on A. pulchella, A. deanei, A. decurrens, and A. mearnsii. In all experiments, the response on prickly acacia could be clearly differentiated from the responses on the non-target species. Both insects were approved for release in Australia. Over a three-year period releases were made at multiple sites in north Queensland, almost all in inland areas. There was no evidence of either insect’s establishment and both colonies were terminated. A new colony of C. assimilis was subsequently established from insects collected in South Africa and releases of C. assimilis from this new colony were made into coastal and inland infestations of prickly acacia. Establishment was rapid at one coastal site and the insect quickly spread to other infestations. Establishment at one inland area was also confirmed in early 2006. The establishment in coastal areas supported a CLIMEX model that indicated that the climate of coastal areas was more suitable than inland areas.     

The potential of Isaria spp. as a bioinsecticide for the biological control of Nasutitermes corniger

The termite Nasutitermes corniger is a serious pest infesting urban areas of Brazil and many other countries. Control largely depends on synthetic pesticides whose indiscriminate use can impact the environment and the health of humans and other animals. Alternative strategies against insect pests, such as biological control by entomopathogenic fungi, could be effective while minimising these deleterious effects. We analysed the actions of the entomopathogenic fungi Isaria farinosa, Isaria fumosorosea, and Isaria javanica against the insect N. corniger. Our results indicated that the fungi examined were pathogenic against N. corniger, with I. farinosa ESALQ1355 being the most efficacious strain, resulting in the death of 95% of the workers (LC₅₀ 6.66?×?10⁴ conidia/mL) and 85% of the soldiers (LC₅₀ 6.81?×?10⁴ conidia/mL). This is the first report of the pathogenicity of Isaria spp. on N. corniger. These in vitro results suggest that I. farinosa ESALQ1355 demonstrates a significant biological potential for controlling N. corniger.     

Efficiency and establishment of three introduced parasitoids of the mealybug Paracoccus marginatus (Hemiptera: Pseudococcidae)

Three introduced parasitoids Acerophagus papayae, Anagyrus loecki, and Pseudleptomastix mexicana of the mealybug Paracoccus marginatus have been released in Miami-Dade and Broward counties (Florida) in 2003. Efficiency and establishment of these previously introduced parasitoids to control P. marginatus were measured in 2005 and 2006, at three locations in Homestead (Miami-Dade). Mealybug populations were initially established on three branches (per plant) of 10 hibiscus plants inside closed-sleeve cages. The three treatments, closed, open, and no-cage environments were applied to the three established mealybug populations on each plant. The number of mealybugs and natural enemies was monitored in all treatments. After 72 h, the number of P. marginatus in the open-sleeve cage was higher than in the no-cage treatment but both were lower than the number in the closed-sleeve cage. Efficiency of parasitoids was evaluated by their percentage parasitism. Percentage parasitism (=recovery) was also used as an indicator for parasitoid establishment. Two of the three previously released parasitoids (A. papayae and A. loecki) were recovered in this study. Acerophagus papayae had a higher percentage parasitism than A. loecki in both the open-sleeve cage (31.0% vs 2.3%) and the no-cage treatments (21.4% vs 1.6%) and caused the most mortality of P. marginatus. There was no recovery of P. mexicana in either of the treatments. Although both A. papayae and A. loecki were established in tested areas, A. papayae was more efficient in controlling P. marginatus than A. loecki. The reasons for not recovering P. mexicana in release areas is yet unknown.     

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5′-TGC GGC TGA GGA GGA CT-3′) and BbasCG1024R (5′-TGC GGC TGA GTG TAG AAC-3′) were designed for detecting and monitoring this isolate in the field.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-012-0292-9) contains supplementary material, which is available to authorized users.     

Genetic analysis of an introduced biological control agent reveals temporal and geographic change,with little evidence of a host mediated shift

The evolution of introduced biological control agents is largely un-explored. Although much is theorized, there is little empirical evidence quantifying the evolutionary dynamics of a biocontrol agent after release into a new environment. In this study we use Diachasmimorpha tryoni, a purposefully introduced biocontrol agent of Ceratitis capitata, to model and quantify spatial, temporal, and host-related evolutionary patterns. This parasitoid has undergone a host shift in its introduced environment, Hawaii, to the gall forming weed biocontrol agent, Eutreta xanthochaeta, an interaction likely mediated by competition for C. capitata with the egg-larval parasitoid Fopius arisanus. To elucidate potential evolutionary patterns we analyzed microsatellites and sequence data extracted from Hawaii and Australian population clusters defined by Structure, in Genepop, Canoco, and IBDWS. Our analysis revealed structuring of Hawaiian D. tryoni populations as defined by significant historic influences related to temporal structure, geographic space, host guild, and augmentative releases. The host-shift parasitoids were not genetically distinct from other Hawaii populations. There were small changes in microsatellite DNA at the population level, but only between Australia and Hawaii populations, not at the host level. These results show that D. tryoni has not undergone host-mediated evolution since introduction to Hawaii, despite the fact that they have expanded their host range in Hawaii to include the gall-forming E. xanthochaeta. To our knowledge this is the first study to quantify genetic differentiation of a biological control agent over geographic space and time using contemporary and museum specimens.