Plant/pathogen interactions observed during host range testing of the rust fungus Uromyces pencanus,a classical biological control agent for Chilean needle grass (Nassella neesiana) in Australia and New Zealand

Nassella neesiana (Chilean needle grass) is a South American grass species that is a serious weed in Australia and New Zealand. The rust fungus Uromyces pencanus is a promising biocontrol agent that could be used to control the weed in both countries. Extensive host range testing has been conducted to explore the specificity of the rust. In this paper we discuss the different degrees of invasion by the rust of the tissues of target and non-target species; the plant defences elicited by such invasion at the cellular level; and their relevance to the biological control of Chilean needle grass.     

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

Detection of the biocontrol agent Colletotrichum coccodes (183088) from the target weed velvetleaf and from soil by strain-specific PCR markers

Diagnostic molecular markers, generated from random amplified polymorphic DNA (RAPD) and used in polymerase chain reaction (PCR), were developed to selectively recognize and detect the presence of a single strain of the biocontrol fungus Colletotrichum coccodes (183088) on the target weed species Abutilon theophrasti and from soil samples. Several isolates of C. coccodes, 15 species of Colletotrichum, a variety of heterogeneous organisms and various plant species were first screened by RAPD-PCR, and a strain specific marker was identified for C. coccodes (183088). No significant sequence similarity was found between this marker and any other sequences in the databases. The marker was converted into a sequence-characterised amplified region (SCAR), and specific primer sets (N5F/N5R, N5Fi/N5Ri) were designed for use in PCR detection assays. The primer sets N5F/N5R and N5Fi/N5Ri each amplified a single product of 617 and 380 bp, respectively, with DNA isolated from strain 183088. The specificity of the primers was confirmed by the absence of amplified products with DNA from other C. coccodes isolates, other species representing 15 phylogenetic groups of the genus Colletotrichum and 11 other organisms. The SCAR primers (N5F/N5R) were successfully used to detect strain 183088 from infected velvetleaf plants but not from seeded greenhouse soil substrate or from soil samples originating from deliberate-released field experiments. The sensitivity of the assay was substantially increased 1000-fold when nested primers (N5Fi/N5Ri) were used in a second PCR run. N5Fi/N5Ri selectively detected strain 183088 from seeded greenhouse soils as well as from deliberate-released field soil samples without any cross-amplification with other soil microorganisms. This rapid PCR assay allows an accurate detection of C. coccodes strain 183088 among a background of soil microorganisms and will be useful for monitoring the biocontrol when released into natural field soils.     

Biological performance of Diclidophlebia smithi (Hemiptera: Psyllidae), a potential biocontrol agent for the invasive weed Miconia calvescens

Diclidophlebia smithi Burckhardt, Morais and Picanço (Hemiptera: Psyllidae) is a promising biological control agent of Miconia calvescens DC. (Melastomataceae), a neotropical invasive weed in forest ecosystems in French Polynesia and Hawaii and a threat in Australia, where it was also introduced. A study on the reproductive performance of D. smithi under laboratory conditions through life expectancy and fertility tables is presented. Results indicated that this psyllid has a high reproductive capacity (R ₀>1 and r _m >0) and a short life cycle (46–47 days) and can have up to nine generations per year. The critical period of its life cycle is during the nymphal stage which is clearly inadequate for field introductions. The best age for introducing D. smithi against M. calvescens is the 4th day of the adult stage. D. smithi is easily mass-reared and has a short life cycle and a high reproductive capacity, which are desirable characteristics for a biological control agent.     

The effects of salinity on the interaction between a pathogen (Listonella anguillarum) and components of a host (Ostrea edulis) immune system

Data are presented from a study to determine how salinity may modulate the interactions between an opportunistic bacterial pathogen Listonella anguillarum and the immune system of a bivalve host, the European flat oyster Ostrea edulis. Oysters were acclimated to three salinity regimes (32, 25 and 16%, at 15 degrees C) for 7 days within the laboratory and were then inoculated with a sub-lethal dose of live L. anguillarum. Forty-eight hours after inoculation measurements were made of the changes in haemocyte composition, haemolymph hydrogen peroxide concentration and haemolymph lysozyme activity to provide information on both the cellular and humoral components of the immune system. The data indicated that in the majority of cases the effects on the immune system were dose dependent. At 32%, a salinity which promoted the growth of the bacterial inoculate, there was a significant increase in the number of circulating large granulocytes and a significant decrease in the haemolymph hydrogen peroxide concentration. At lower salinities, which were less favourable to the growth of L. anguillarum, there were no significant immune system effects. The data highlight the potential for environment management as a tool in controlling opportunistic pathogens and subsequently disease in commercially important bivalve species.     

Biology and host range of Ophiomyia camarae Spencer (Diptera: Agromyzidae), a potential biocontrol agent for Lantana spp. (Verbenaceae) in Australia

The life history and host range of the herringbone leaf-mining fly Ophiomyia camarae, a potential biological control agent for Lantana spp., were investigated. Eggs were deposited singly on the underside of leaves. Although several eggs can be laid on a single leaf and a maximum of three individual mines were seen on a single leaf, only one pupa per leaf ever developed. The generation time (egg to adult) was about 38 days. Females (mean 14 days) lived longer than males (mean 9 days) and produced about 61 mines. Oviposition and larval development occurred on all five lantana phenotypes tested. Eleven plant species representing six families were tested to determine the host range. Oviposition and larval development occurred on only lantana and another nonnative plant Lippia alba (Verbenaceae), with both species supporting populations over several generations. A CLIMEX model showed that most of the coastal areas of eastern Australia south to 30°16′ S (Coffs Harbour) would be suitable for O. camarae. O. camarae was approved for release in Australia in October 2007 and mines have been observed on plants at numerous field sites along the coast following releases.     

Free fatty acids from the weed,Polygonum orientale leaves for attraction of the potential biocontrol agent,Galerucella placida (Coleoptera: Chrysomelidae)

Extraction, thin layer chromatography and gas chromatography-mass spectrometry of leaf surface waxes of Polygonum orientale L. (Polygonaceae) weed revealed 11, 15 and 11 free fatty acids in young, mature and senescent stages. Oleic acid was the predominant in young leaves (5950 ± 111 µg); whereas palmitic acid was the predominant fatty acids, representing 4247.5 ± 23 and 6644 ± 110 µg in mature and senescent leaves, respectively. Both tridecanoic and heneicosanoic acids were not detected in young and senescent leaves, and myristic and heptadecanoic acids were not identified in young leaves; whereas lauric and nonadecanoic acids were not detected in senescent leaves. The free fatty acids from young, mature and senescent weed leaves, and the mixtures of synthetic fatty acids mimicking free fatty acids of three types of weed leaves attracted female Galerucella placida (Coleoptera: Chrysomelidae) at the minimal amounts of 2, 1 and 2 leaf equivalent free fatty acids, respectively, in Y-shaped glass tube olfactometer bioassays under laboratory conditions. Individual synthetic pentadecanoic, palmitoleic, stearic, nonadecanoic and docosanoic acids at 44.82, 9.91, 92.22, 18.33 and 15.88 µg, respectively, elicited attraction of the insect. A synthetic blend of 3.59, 7.89, 44.82, 9.91, 32.31, 18.33 and 15.88 µg of lauric, myristic, pentadecanoic, palmitoleic, heptadecanoic, nonadecanoic and docosanoic acids, respectively, indicated highest attraction of the insect.     

Modeling the impact of a biocontrol agent, Puccinia lagenophorae, on interactions between a crop, Daucus carota, and a weed, Senecio vulgaris

The impact of a biocontrol agent spreading from a point source on crop–weed interactions was modeled. The model encompassed: (i) severity of crop–weed competition as affected by a rust pathogen, (ii) velocity of spread of the rust pathogen, and (iii) density of infected plants introduced in the weed population as starting points (inoculum sources) for an epidemic. The model was parameterized for a study system encompassing the crop Daucus carota (carrot), the weed Senecio vulgaris (common groundsel), and its antagonist Puccinia lagenophorae. The parameters of (i) were estimated in a greenhouse study using a response-surface design. Estimates of the parameter of (ii) were obtained from the literature. The density of infected plants (iii) was varied to simulate crop loss as function of density. Simulations were run for various densities of the weed and various velocities of rust pathogen spread. The results of the simulations indicated a crop-loss ranging from 5 to 10% at levels of relatively weak D. carota–S. vulgaris competition. Density of inoculum sources and velocity of P. lagenophorae spread had only minor effects on crop loss. In contrast, density of inoculum sources and velocity of spread had major effects on crop loss at levels of intermediate (range of 10–35% loss) and severe competition (range of 30–70% loss). The results are discussed both with respect to biological control of S. vulgaris using P. lagenophorae as biocontrol agent and as a general model describing the impact of the spatial dynamics of a pathogen (natural enemy) on plant competition.     

Population dynamics of Spalgis epius (Lepidoptera: Lycaenidae), a candidate biocontrol agent of mealybugs and its interaction with mealybug-attendant ants

Spalgis epius is an economically important hemipterophagous butterfly. Detailed information on the population dynamics, natural enemies, and prey range of S. epius and its association with mealybug-attendant ant species is lacking. Three years of field studies conducted at Bangalore University campus, Bengaluru, India on these aspects indicated that the population density of S. epius was greatest from June to December and least from February to May in the low land region. S. epius survived on eight prey species, which were present in different months, of which Phenacoccus indicus was recorded as a prey of S. epius for the first time. Different prey species occurred on 12 species of host plants. The occurrence of S. epius was negatively correlated with temperature and positively correlated with relative humidity and prey populations. Six mealybug-attendant ant species were associated with the larvae of S. epius. The seven general predators of S. epius adults were recorded. Knowledge on the population dynamics and a prey range of S. epius and its interaction with mealybug-attendant ant species could be helpful to using this predator as a major biocontrol agent of various species of mealybugs. This study contributed to our understanding of the population dynamics of a hemipterophagous butterfly.     

Biology and host preferences of Cryptorhynchus melastomae (Coleoptera: Curculionidae), a possible biocontrol agent for Miconia calvescens (Melastomataceae) in Hawaii

The introduced plant Miconia calvescens (Melastomataceae) poses a grave threat to Hawaii's native ecosystems and biodiversity. One potential candidate for classical biological control is Cryptorhynchus melastomae (Coleoptera: Curculionidae: Cryptorhynchinae), a stem-boring weevil from Central and South America. This weevil feeds on M. calvescens in its native Costa Rica and has been successfully reared under greenhouse conditions. Comparison of its environmental conditions in Costa Rica with those in the Miconia infested areas of Hawaii indicates the latter is a suitable habitat for C. melastomae. C. melastomae has one or two generations per year. Adults feed on new stems, petioles, leaf buds, veins, and lamina, whereas larvae mine the stem until pupation. Adults appear to prefer saplings for oviposition and feeding. Under greenhouse conditions both adults and larvae can seriously damage and kill small M. calvescens. Preliminary host testing indicates that C. melastomae may be family specific on Melastomataceae. However, because Hawaii lacks native melastomes and has many other serious melastome weeds, a family specific insect may be suitable as a biocontrol agent in this case.     

Coevolution of a homing endonuclease and its host target sequence

We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.     

Structural analysis of the interaction between urokinase-type plasminogen activator and its receptor: a potential target for anti-invasive cancer therapy

The ability to degrade the extracellular matrix by controlled proteolysis is an important property of malignant cancer cells, which enables them to invade the surrounding tissue and to gain access to the circulation by intravasation. One proteolytic system thought to be involved in these processes is urokinase-mediated plasminogen activation. Expression of a glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) targets this system to the cell surface. This receptor (uPAR) is composed of three homologous modules belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Integrity of the three-domain structure of uPAR is required for maintenance of its sub-nanomolar affinity for uPA, but the functional epitope for this interaction is primarily located in uPAR domain I. Using affinity maturation by combinatorial chemistry, we have recently identified a potent 9-mer peptide antagonist of the uPA-uPAR interaction having a high affinity for uPAR (K(d)< 1 nM). Photoaffinity labelling suggests that this peptide interacts with a composite binding site in uPAR involving both domains I and III. When tested in a chicken chorioallantoic membrane assay that was developed to quantify intravasation of human cells, this antagonist was able to reduce the intravasation of HEp-3 cancer cells by approx. 60%.     

Studies on the rust,Maravalia cryptostegiae,a potential biological control agent of rubber-vine weed,Cryptostegia grandiflora (Asclepiadaceae: Periplocoideae), in Australia,II: Infection

The parameters which govern infection of rubber-vine weed by the rustMaravalia cryptostegiae were investigated. The infection process, from appressorial formation to sporulation, is described and illustrated. Uredinioid teliospores have an optimum temperature range for germination at 22–27 °C, both in vitro and in vivo. However, germination on the rubber-vine leaf was more than double (81–92%) that in the absence of the host, and appressoria were formed only in vivo. An optimum temperature of 20–22°C and a dew period of 12 hours or more gave the highest level of infection as measured by sporulation density. The latent period from inoculation to pustule formation decreased with increasing temperature; the shortest period (8–11 days) being recorded at 25–27°C. At the lower temperatures (18°C), this was significantly extended (19–21 days). Four successive inoculations significantly reduced plant height and dry weight, although a compensatory growth flush occurred after the third inoculation. The addition of cryoprotectants had a negative affect on spore viability and subsequent infectivity. Cooling dry spores to –196°C at the rate of 10°C min^–1 gave the best results, with high germination (93–65%) up to 8 days after thawing.     

Biomechanical interaction between hyphae of two Pythium species (Oomycota) and host tissues

Forces exerted by hyphae of the phytopathogen Pythium graminicola and mammalian pathogen Pythium insidiosum were compared with the mechanical resistance of their hosts' tissues. Hyphal apices of both species exerted a mean force of 2 microN, corresponding to mean pressures of 0.19 microN microm(-2) (or MPa) for P. graminicola, and 0.14 microN microm(-2) for P. insidiosum. Experiments with glass microprobes showed that the epidermis of grass roots resisted penetration until the pressure applied at the probe tip reached 1-12 microN microm(-2). Previously published data show that mammalian skin offers even greater resistance (10-47 microN microm(-2)). Clearly, tissue strength exceeds the pressures exerted by hyphae of these pathogens, verifying that secreted enzymes must play a critical role in reducing the resistance of plant and animal tissues. It is presumed that hyphae are sufficiently powerful to bore through any obstacles remaining after enzyme action.     

Performance and impact of the biological control agent Xubida infusella (Lepidoptera; Pyralidae) on the target weed Eichhornia crassipes (waterhyacinth) and on a non-target plant, Pontederia cordata (pickerelweed) in two nutrient regimes

Xubida infusella (Walker) (Lepidoptera: Pyralidae) is potentially a useful biological control agent targeting Eichhornia crassipes (waterhyacinth) in the USA but many regions infested with waterhyacinth are also inhabited by an alternative native host, Pontederia cordata (pickerelweed). Experiments were conducted in Australia to assess the impact of X. infusella on pickerelweed compared to waterhyacinth where both these plants were available and X. infusella had already been released. Overall X. infusella had a greater impact on pickerelweed than on waterhyacinth. More than one larva per plant was required to reduce the total shoot dry weight of waterhyacinth but only one larva per plant reduced the total shoot dry weight of pickerelweed. Insect feeding caused the number of secondary shoots (daughter plants) of pickerelweed to double whereas the number of daughter plants produced by waterhyacinth remained unchanged. We suggest this indicates a considerable impact on pickerelweed rather than effective compensation for insect damage because the shoots produced were very small. Waterhyacinth produced a constant number of daughter plants when fed on by up to three larvae per plant. Higher nitrogen status of both species of host plant increased the rate of larval development and pupal weight of X. infusella. The weight and fecundity of X. infusella reared on pickerelweed were lower than those reared on waterhyacinth but large numbers of progeny were produced on both plant species. This experiment demonstrates a considerable impact of X. infusella on pickerelweed suggesting this plant is at risk from this agent if released in the USA where pickerelweed is present. The considerable impact on waterhyacinth demonstrates the potential for this insect to contribute to waterhyacinth control in countries where risk assessment favours release.     

Gratiana boliviana, a potential biocontrol agent of Solanum viarum: Quarantine host-specificity testing in Florida and field surveys in South America

Host-specificity tests andfield surveys were conducted to determine thesuitability of the tortoise beetleGratiana boliviana Spaeth (Coleoptera:Chrysomelidae) from Argentina for classicalbiological control of Solanum viarumDunal (Solanaceae) in the USA. Thehost-specificity tests were conducted at theFlorida Department of Agriculture and ConsumerServices-Division of Plant Industry quarantinefacility in Gainesville, Florida (USA). Multiple-choice host-specificity tests wereconducted in small cages using 123 plantspecies in 35 families. Adults fedsignificantly (>41% of the leaf area offeredwas damaged) on the target weed(S. viarum), and fed lightly (<20%) onSolanum torvum Sw. (noxious weed native towest-Africa). Adults did some exploratoryfeeding (<5%) on eggplant, Solanummelongena L. (economic crop), Solanumelaeagnifolium Cav. (major agricultural weedin the western US), and on Solanumtampicense Dunal (weed of Central Americanorigin). No feeding was observed on any of theother 118 plant species that were testedincluding another 21 Solanum species.G. boliviana adults laid an average of 68eggs per female on S. viarum, 5 eggs perfemale on S. torvum, and an average of0.2 eggs on eggplant. No-choicehost-specificity tests were also conducted inwhich G. boliviana adults and neonatelarvae were exposed to 19 and 22 plant speciesrespectively. Tests with the neonatesindicated this insect was able to complete itsdevelopment only on S. viarum (67%reached the pupae stage). The no-choice testswith adults indicated that this insect fed,laid eggs, and completed development only onS. viarum. The unsprayed eggplant fieldsthat were surveyed in its natural range inArgentina, Brazil, Paraguay, and Uruguay fromJune 1997 to March 2000 indicated thatG. boliviana is not a pest of eggplants in SouthAmerica. Therefore, a petition for fieldrelease of G. boliviana for classicalbiological control of S. viarum in theUSA was submitted in April 2000.     

Interactions between Oncopera alboguttata (Lepidoptera: Hepialidae) and its microsporidan pathogen, Pleistophora oncoperae (Protozoa: Microsporida)

Infection by Pleistophora oncoperae is widespread in populations of Oncopera alboguttata. Detailed study of insects from one site indicated that 85% of the 131 pupae and 65% of the 403 adults were infected. The infection did not adversely affect duration of larval and pupal development, adult life span, the number of eggs laid, or fecundity. Infected females were able to transmit the disease to their progeny, though the degree of transmission ranged from 0 to 92%, depending on the severity of the parental infection. Transmission of the disease by infected males could not be detected. It was estimated that 49% of the progeny from this site would have been infected by transovum transmission. Details of the incidence of P. oncoperae infection from this area during the period 1972 to 1979 are given to emphasise the possible role of this disease in O. alboguttata population dynamics.     

Biology, host range, and risk assessment supporting release in Africa of Falconia intermedia (Heteroptera: Miridae), a new biocontrol agent for Lantana camara

The ornamental hybrid shrub, Lantana camara L. (lantana), is a serious environmental weed and has been targeted for biological control in South Africa since 1961. The established biocontrol agents cause insufficient levels of damage and additional natural enemies are required to reduce the invasiveness of this weed. The lantana mirid, Falconia intermedia (Distant), is a promising new agent that was imported from the Caribbean for life history and host-range studies. The nymphs and adults are leaf-suckers that cause chlorotic speckling, which reduces the photosynthetic capacity of the plant. Biological studies indicate that F. intermedia has considerable biocontrol potential, in that it has a high intrinsic rate of increase, the potential for multiple generations a year, highly mobile adults, and a high level of damage per individual. Host-specificity trials indicated that the lantana mirid has a narrow host range, with L. camara being the most suitable host, but several indigenous African species in the closely related genus Lippia are suitable alternative host plants. Under multiple-choice conditions, adults showed a significant and strong oviposition preference for L. camara over the Lippia species. A risk assessment of potential nontarget effects indicated that three Lippia species could sustain damage levels in the field. The relatively low probability of damage to indigenous species was considered a justified trade-off for the potentially marked impact on L. camara. The regulatory authorities accepted the results of this study and F. intermedia was released against L. camara in South Africa in April 1999.