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.     

The fungus Phoma multirostrata is a host-specific pathogen and a potential biocontrol agent for a broadleaf weed

Since the increasing prevalence of herbicide-resistant weeds and herbicide bans, the use of biological controls with mycoherbicides become an innovative approach of weed control. In this study, we verified the pathogenicity of Phoma multirostrata TBRC 12769 against the common weed in Thailand, tridax daisy (Tridax procumbens), with its mechanism of infection unveiled by fluorescence microscopy. P. multirostrata directly penetrated through epidermal cells, stomata, and trichomes at 48 h post-inoculation. The hyphae also propagated in the lumen of the trichome, enabling the fungus to grow subcuticular to neighboring weed tissues at the bases of leaf trichomes. The necrotic pattern emerged around the trichome. During necrosis, unicellular chlamydospores were also detected inside the leaf trichomes, suggesting an overwintering stage under stress and nutrient-depleting conditions. Trichomes of weed leaves were found to be key infection sites for pathogenesis. Topical application of conidial suspension on T. procumbens potted plants led to 60–98% and 65 and 87% disease incidence under laboratory and greenhouse conditions, respectively, on days 15–20 post-inoculation. The 16-h dew period incubation results in a sharp increase by 37% in the pathogenicity rate. The greenhouse trials verified that the fungus is non-pathogenic to eight crops. Our LC-MS analysis indicated that norharman, a known bioherbicidal compound, and other compounds were detected in the supernatant fraction of fungal culture, of which resulted in a blight symptom on T. procumbens leaves. This study demonstrated that the P. multirostrata isolate is an effective mycoherbicide for this broadleaf weed.     

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.     

Factors affecting the efficacy of the leaf-spot fungus Mycosphaerella polygoni-cuspidati (Ascomycota): A potential classical biological control agent of the invasive alien weed Fallopia japonica (Polygonaceae) in the UK

Fallopia japonica, commonly known as Japanese knotweed, is an increasingly serious invasive alien weed in the UK and large parts of mainland Europe, as well as in North America. There is an urgent need to include classical biological control (CBC) into any integrated pest management strategy. The leaf-spot pathogen Mycosphaerella polygoni-cuspidati, a coevolved natural enemy of F. japonica present throughout its native Japanese range, is considered to have high potential as a CBC agent. In this study, the disease development of M. polygoni-cuspidati in the field and the optimum infection parameters under controlled conditions were investigated to elucidate the pathogen’s potential biocontrol efficacy against Japanese knotweed. Field observation in Japan showed that M. polygoni-cuspidati caused severe damage to its host plant. When sentinel knotweed plants from the UK were placed amongst naturally-infected field populations of F. japonica, disease incidence and severity were highest in July when monthly precipitation was also highest. In greenhouse inoculation tests, F. japonica was shown to be most susceptible at the young leaf stage (7–12 days after opening). Disease severity was highest after an initial dew period of 42–48 h, and severe defoliation followed inoculation at a temperature range of 15–25 °C. The optimum post-inoculation temperature after dew treatment for disease severity was 20–25 °C. In field inoculation tests, high disease incidence and severity indicate that the pathogen has the potential to control the plant effectively in the field. Humidity and temperature were shown to be the main factors influencing disease expression and lesion development of M. polygoni-cuspidati in a field situation. These results provide valuable information for any future use of M. polygoni-cuspidati as a CBC agent for management of Japanese knotweed in the UK.     

Pollinator-mediated interactions between a pathogenic fungus, Uromyces pisi (Pucciniaceae), and its host plant,Euphorbia cyparissias (Euphorbiaceae)

The plant Euphorbia cyparissias is commonly infected by rust fungi of the species complex Uromyces pisi. When infected, E. cyparissias is unable to flower, but instead is induced by the fungus to form pseudoflowers. Pseudoflowers are rosettes of yellow leaves upon which the fungus presents its gametes in a sweet-smelling fungal nectar. We hypothesized that the fungi, as they are heterothallic, are dependent on insect visitation to cross-fertilize their mating types. We confirmed that insects are required with an insect exclusion experiment. We further hypothesized that pseudoflowers of U. pisi interact with uninfected true host flowers through insects during their period of co-"flowering" in early spring. We conducted artificial array experiments in the field to test whether the two species share insects and whether they influenced each other's insect visitation. Insects moved between true flowers and pseudoflowers, but true flowers received more visits over all. Pseudoflowers and true flowers did not influence each other's visitation rates in mixtures. However, shorter visits were observed on pseudoflowers in mixtures than monocultures, suggesting that true flowers might be competitors for pseudoflowers. Further experiments are needed to determine whether the similarity of pseudoflowers to true flowers is adaptive.     

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.     

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.     

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.     

Effects of a biocontrol agent of apple powdery mildew (Podosphaera leucotricha) on the host plant and on non-target organisms: an insect pest (Cydia pomonella) and a pathogen (Venturia inaequalis)

A number of studies have focused on the selection and use of new biocontrol agents, but the effects of the introduction of these microorganisms on non-target organisms, including the crop plants themselves, are not well known. Non-target effects of sprayed applications of a potential biocontrol agent of apple powdery mildew (Podosphaera leucotricha Ell. Et Ev.), on scab infections (Venturia inaequalis Cooke Winter), on codling moth [Cydia pomonella L. (Lepidoptera: Tortricidae)] oviposition and damage and apple (Malus x domestica) fruit quality are examined. This biocontrol agent, an epiphytic yeast isolate called Y16, affected neither conidia germination of V. inaequalis nor their penetration of the leaf tissue but suppressed the disease caused by this pathogen. The quantity of eggs laid by the codling moth during its second flight period on yeast treated trees was significantly different to the quantity of eggs laid on the untreated trees. In the first season of the experiments, more eggs were laid on the treated trees, especially on those tree parts closest to the fruit. These results, however, were not confirmed the following season: fewer eggs were laid on the treated trees than on the untreated trees. These conflicting observations are attributed to year-to-year variation in environmental conditions, which may affect yeast survival and activity. A 2-month-long assay was conducted in the orchard during the codling moth's second flight period from mid-July until mid-September. The yeast treatment did not affect the damage caused by the codling moth to the fruits. Finally, the yeast treatment did not affect any of the examined fruit quality parameters.     

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.     

入侵植物水花生解剖学和组织化学染色研究

水花生(Alternanthera philoxeroides)因其表型可塑性、高生长速率和快速无性繁殖能适应水、陆生境.该文利用光学显微镜和荧光显微镜对水、陆生境的水花生不定根、茎解剖结构、组织化学特征及质外体通透性进行了研究.结果表明:(1)水生境下,其不定根皮层中具较大裂生型通气组织,无次生生长,内皮层具凯氏带且...     

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.     

Antifungal activity of puroindoline protein from soft wheat against grain molds and its potential as a biocontrol agent

Mold growth reduces the quality of stored grains, besides producing toxins that pose a potential threat to human health. Therefore, prevention of grain mold growth during storage is important to ensure a safe and high-quality product, preferably using an eco-friendly antifungal agent. The puroindoline (PIN) protein was extracted by Triton X-114 and identified by QE mass spectrometry. Aspergillus flavus has attracted much attention because of its toxic secondary metabolites, and PIN protein showed a significant inhibition on A. flavus growth. Scanning electron microscopy revealed altered spore morphology of A. flavus following PIN protein treatment, and propidium iodide staining showed incomplete spore cell membranes. The disruption and deformation of A. flavus spores suggest that the cell walls and cell membranes were compromised. Decreased mitochondrial membrane potential and increased levels of intracellular reactive oxygen specieswere detected using JC-1 and 2,7-dichlorodihydrofluorescein diacetate staining, respectively. PIN protein could effectively inhibit the growth and aflatoxins B1 production of A. flavus in stored grains, such as wheat and rice. PIN proteins can inhibit the growth of many common grain storage molds, including Penicillium, Aspergillus spp. (A. flavus, A. glaucus, A. kawachii, A. ochraceus and A. niger), Alternaria and Fusarium graminearum, in a dose-dependent manner. PIN protein has a significant inhibitory effect on the growth of grain molds, with a stronger inhibitory effect noted in wheat and rice. Our study provides a novel and simple theoretical basis for the selection and storage of mold resistance in grains and food during storage.     

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.     

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis-related (PR). An increase in abundance of PR-3 or PR-5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR-proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.     

Laboratory host range testing of Neomusotima conspurcatalis (Lepidoptera: Crambidae) – a potential biological control agent of the invasive weed,Old World climbing fern,Lygodium microphyllum (Lygodiaceae)

Old World climbing fern, Lygodium microphyllum, is a serious invasive weed in south Florida. Development of biological control is vital for sustainable management of L. microphyllum. Neomusotima conspurcatalis was discovered in Hong Kong in 1997 and was subsequently found causing feeding damage on L. microphyllum in much of its native range in Asia. Quarantine testing of N. conspurcatalis used 37 non-Lygodium fern species representative of New World genera of cultivated ferns and fern allies, one gymnosperm, three crop species, six Lygodium species, and the primary host L. microphyllum. No significant oviposition or feeding was observed on any of the 41 non-Lygodium species evaluated. Oviposition and feeding occurred on all Lygodium species, but amounts were low and usually significantly less than observed on L. microphyllum. The exception was L. japonicum, which was preferred as an oviposition host. Neomusotima conspurcatalis was only able to complete development on L. japonicum and L. palmatum, but survival on these species was only half that occurring on L. microphyllum. Neomusotima conspurcatalis is a Lygodium specialist. Lygodium japonicum is an invasive weed in the United States. Lygodium palmatum is restricted to areas of the United States where freezing winter temperatures would be lethal to N. conspurcatalis. It was concluded that N. conspurcatalis would pose no threat to native or cultivated plants in North America or the Caribbean and should be considered for biocontrol of L. microphyllum. A release petition was submitted in 2005. An USDA-APHIS release permit for N. conspurcatalis was issued in 2007.