Establishment, population increase, spread, and ecological host range of Lophodiplosis trifida (Diptera: Cecidomyiidae), a biological control agent of the invasive tree Melaleuca quinquenervia (Myrtales: Myrtaceae).

The Australian tree Melaleuca quinquenervia (Cavanilles) Blake is an invasive weed in wetland systems of Florida. A biological control program targeting M. quinquenervia has resulted in the release of the gall forming midge Lophodiplosis trifida Gagné (Diptera: Cecidomyiidae). Populations of the introduced herbivore readily established at all 24 release sites across the weed's range in Florida, and there was no evidence that founding colony size (100, 2,000, or 6,000 adults) influenced herbivore establishment or local population growth rates. Landscape level spread of L. trifida from release sites averaged nearly 6 km/yr, ranging as high as 14.4 km/yr. Prerelease host range testing predicted that L. trifida oviposits indiscriminately on test plant species but does not complete development on any of the test species, including congeners present in Florida. To test the predictability of these host range tests, L. trifida was released in a common garden consisting of 18 test plant species that were interplanted with M. quinquenervia. Plant species postulated to be at risk experienced no gall development by L. trifida while intermingled M. quinquenervia trees supported 704.8 (± 158.5) galls per plant. Historically, many introduced Cecidomyiidae have limited effect on plant performance of target weeds because of recruitment of native parasitoids that disrupt biological control efficacy. In contrast to this trend, there has been no evidence to date that parasitoids are exploiting L. trifida in Florida.

First Report of Puccinia psidii Caused Rust Disease Epiphytotic on the Invasive Shrub Rhodomyrtus tomentosa in Florida.

Rhodomyrtus tomentosa (Aiton) Hassk. (downy-rose myrtle, family: Myrtaceae), of South Asian origin, is an invasive shrub that has formed monotypic stands in Florida (3). During the winter and spring of 2010 through 2012, a rust disease of epiphytotic proportion was observed on young foliage, stem terminals, and immature fruits of this shrub in natural areas of Martin and Lee counties, Florida. Expanding leaves and succulent stems developed chlorotic flecks on the surface that developed into pustules and ruptured to discharge urediniospores. Symptomatic leaves and stems developed severe necrotic spots and resulted in tissue distortion, defoliation, and stem dieback. Based on symptoms and urediniospore morphology and dimensions (17.7 to 26.1 [22.1 ± 0.3] × 14.7 to 21.1 [17.7 ± 0.2] µm; n = 51) (4), the causal agent was identified as Puccinia psidii Winter; teliospores were not observed in samples since it does not produce these spore stages below 20°C ambient temperature (1). This identification was confirmed by a GenBank BLAST of internal transcribed spacer (ITS) sequences (Accession Nos. KC607876 and KC607877) that showed 99% identity with 42 sequences of P. psidii from diverse host species and locations. P. psidii is believed to be of neotropical origin and has a host range of 129 species in 33 genera within Myrtaceae (2). However, P. psidii caused disease of downy-rose myrtle has not been previously reported in Florida, even though severe infections occurred on another invasive tree, Melaleuca quinquenervia (Cav.) S.F. Blake (3), growing in adjacent areas. In December 2011, urediniospores were collected from downy-rose myrtle, established in aqueous suspension (45,000 spores/ml), and spray inoculated on potted downy-rose myrtle plants (n = 3), which were maintained in 100% ambient humidity, at 20°C, with a 12-h light cycle for 72 h. Plants mock-inoculated with water served as the negative control. Disease symptoms, including chlorotic flecks and raised surfaces, appeared on leaf lamina in 3 to 6 days on P. psidii-inoculated plants, while control plants remained symptomless. Raised surfaces developed into distinct pustules and eventually erupted to discharge urediniospores within 6 to 12 days of inoculation. Tests were repeated once during March and April of 2012 with the same results. The latent and incubation periods reported herein are within the previously reported range for P. psidii (2,4). To our knowledge, this is the first confirmed report of P. psidii epiphytotic on downy-rose myrtle populations in Florida. The recent occurrence of P. psidii epiphytotic on downy-rose myrtle raises critical questions as to why this myrtle rust disease is so severe and widespread on this host after decades of presumed exposure to P. psidii in Florida. Because this rust pathogen has emerged as a major invasive threat to many myrtaceous species around the world, further genotyping and cross-inoculation studies are needed to determine the host specificity and potential origin of the P. psidii isolates derived from downy-rose myrtle (2). References: (1) A. C. Alfenas et al. Australas. Plant Pathol. 32:325, 2003. (2) A. J. Carnegie and J. R. Lidbetter. Australas. Plant Pathol. 41:13, 2012. (3) K. A. Langeland and C. K Burks, eds. Identification and biology of non-native plants in Florida's natural areas. University of Florida, Gainesville, 1998. (4) M. B. Rayachhetry et al. Biol. Contr. 22:38, 2001.

Differential Response by Melaleuca quinquenervia Trees to Attack by the Rust Fungus Puccinia psidii in Florida.

Melaleuca quinquenervia (melaleuca) is an exotic invasive tree in Florida, Hawaii, and some Caribbean islands (1,2). Puccinia psidii (rust fungus) attacks melaleuca as well as other plants in a few genera of the Myrtaceae and Heteropyxidaceae, both members of the Myrtales (1,2). Disease occurs on succulent stems and foliage of melaleuca, causing twig dieback and defoliation (3). Melaleuca trees growing under similar field conditions exhibit susceptible or resistant reactions toward this fungus. To document this differential susceptibility of melaleuca to P. psidii, we visually evaluated 331 field-grown melaleuca trees from southeast Florida for occurrence of disease attributes: pustules (susceptible), nonpersistent halos (resistant), or asymptomatic (no macroscopic symptoms) conditions on leaves and succulent twigs during February and March when symptoms were at their peak. Percentages of trees manifesting susceptible, resistant, and asymptomatic responses to this fungus were 85.8, 13.0, and 1.2%, respectively. A screenhouse study was conducted to corroborate these observations by raising plants from composite seed sources and maintaining them in seven 3.8-liter plastic pots that were filled with commercial potting media. Nine to eleven plants per pot (with new foliage) were individually tagged, grown to 30 to 45 cm high, and spray inoculated (during February and March) with uredospores (~2 × 106/ml) obtained from melaleuca trees and suspended in water. Inoculated plants were placed on a screenhouse bench under infected trees and subjected to additional inoculum, thereby simulating field conditions. Evaluations made weekly during a 4-week period revealed that susceptible, resistant, and asymptomatic seedlings constituted 63.3, 33.6, and 3.2%, respectively, of the tagged plants. To assess the stability of these fungal and host attributes over time and space, we multiplied two P. psidii susceptible and two resistant plants from cuttings. We spray inoculated 6 to 13 rooted cuttings from each plant types with uredospores (0.8 to 2 × 106/ml) obtained from diseased melaleuca trees and suspended in water. These plants were incubated in a dew chamber for 72 to 96 h under 100% relative humidity at 19 to 23°C maintained with a 12-h fluorescent light cycle. After incubation, plants were placed randomly on a bench in a screenhouse (21 to 23°C) and evaluated weekly for symptom development during a 4-week experimental period. Noninoculated controls were maintained as well. The experiment was repeated twice. Foliage of the resistant plants developed a few incipient halos whereas 100% of the susceptible plants developed erupted uredinia and were defoliated in both replications. No detectable change in P. psidii virulence and melaleuca susceptibility patterns was observed. Despite wide host range within Myrtales, resistance to P. psidii exists within M. quinquenervia. Other P. psidii susceptible host systems of economic and environmental importance may have host/pathogen relationships similar to that of melaleuca and the selection of resistant individuals from their affected populations may be possible. Additional studies will be needed to ascertain the attributes of virulence or resistance in this rust fungus-melaleuca association. References: (1) M. Glen et al. Australas. Plant Pathol. 36:1, 2007. (2) P. D. Pratt et al. J. Aquat. Plant Manag. 45:8, 2007. (3) M. B. Rayachhetry et al. Biol. Control 22:38, 2001.

Geographic distribution and regional impacts of Oxyops vitiosa (Coleoptera: Curculionidae) and Boreioglycaspis melaleucae (Hemiptera: Psyllidae), biological control agents of the invasive tree Melaleuca quinquenervia.

The invasive tree Melaleuca quinquenervia (Cav.) Blake is widely distributed throughout peninsular Florida and poses a significant threat to species diversity in the wetland systems of the Everglades. Mitigation of this threat includes the areawide release campaign of the biological control agents Oxyops vitiosa Pascoe and Boreioglycaspis melaleucae Moore. We summarize the results of this release effort and quantify the resulting geographic distribution of the herbivores as well as their regional impact on the target weed. A combined total of 3.3 million individual Melaleuca biological control agents have been redistributed to 407 locations and among 15 Florida counties. Surveys of the invaded area indicate that the geographic distribution of O. vitiosa encompasses 71% of the Melaleuca infestation. Although released 5 yr later, the distribution of B. melaleuca is slightly greater than its predecessor, with a range including 78% of the sampled Melaleuca stands. Melaleuca stands outside both biological control agents' distributions occurred primarily in the northern extremes of the tree's range. Strong positive association between herbivore species was observed, with the same density of both species occurring in 162 stands and no evidence of interspecific competition. Soil type also influenced the incidence of biological control agents and the distribution of their impacts. The odds of encountering O. vitiosa or B. melaleucae in cells dominated by sandy soils were 2.2 and 2.9 times more likely than those predominated by organically rich soils. As a result, a greater level of damage from both herbivores was observed for stands growing on sandy versus organic-rich soils.

First Report of Infection of Lygodium microphyllum by Puccinia lygodii, a Potential Biocontrol Agent of an Invasive Fern in Florida.

Lygodium microphyllum (Cav.) R.Br. (Old World climbing fern), in the family Schizaeaceae, is one of the most invasive (Category I in Florida) weeds in Florida. It has invaded more than 50,000 ha of wetlands and moist habitats in southern Florida and is rapidly spreading in new areas of the Everglades (3). The search and evaluation of biocontrol agents for this fern is currently in progress. Puccinia lygodii (Har.) Arth. (Uredinales) (1), previously recorded on L. volubile Sw. and L. venustum Sw. in South America (2), attacks foliage and severely damages L. japonicum Thunb. (Japanese climbing fern) vines in northern and central Florida (4). We hypothesized that since L. japonicum occurred mainly in northern and central Florida, P. lygodii did not have opportunity to interact with L. microphyllum, which primarily occurs in southern Florida. Therefore, we used two inoculation methods to test the possible pathogenicity of P. lygodii on the new host, L. microphyllum. Method-I was designed to imitate a seminatural inoculation technique in which three containerized (0.45-L capacity) L. microphyllum test plants (15- to 30-cm-high sporelings) were intermixed among a group of containerized (5.0-L capacity) P. lygodii-infected L. japonicum plants (source of inoculum) in a glasshouse. In Method-II, uredospores obtained from pustules on diseased L. japonicum foliage were adjusted to 1 × 106 uredospores/ml and then misted on three L. microphyllum sporelings (same size as in Method-I) until foliage was completely wet. The plants were then covered individually with a plastic bag for 3 days to facilitate spore germination and infection. In both methods, three L. japonicum sporelings of similar size as L. microphyllum were intermixed among diseased L. japonicum plants as a positive control. All test and infected plants were placed on 6-cm-high trays filled two-thirds with water and exposed to diffused daylight and a temperature range of 20 to 35°C in a glasshouse. These plants were monitored for the development of rust symptoms (halos and rust pustules) development for 8 weeks. Minute cinnamon flakes that developed into eruptive pustules were seen on the lower surface of the pinnules approximately 42 and 28 days after treatment initiation (in both methods) for L. microphyllum and L. japonicum (positive control), respectively. Each method was repeated twice. Dimensions (29.7 [±3.7] × 23.5 [±2.6] µm) and morphology of urediniospores from pustules on inoculated L. microphyllum were similar to those reported for P. lygodii on other host systems (1,2,4). To our knowledge, this is the first report demonstrating the infection of P. lygodii on L. microphyllum. The potential use of P. lygodii as a classical bio-control agent of L. microphyllum in southern Florida will be further investigated. References: (1) J. C. Arthur. Bull. Torrey Bot. Club 51:55, 1924. (2) J. W. McCain et al. Mycotaxon 39:281, 1990. (3) R. W. Pemberton. SIDA 20:1759, 2003. (4) M. B. Rayachhetry et al. Plant Dis. 85:232, 2000.

Low-density releases of Neoseiulus fallacis provide for rapid dispersal and control of Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae) on apple seedlings.

Releases of Neoseiulus fallacis (Garman) at 1500--6000 per ha when prey were at 0.1-0.3 per leaf provided seasonal control of Tetranychus urticae Koch (all stages) at 1-2 per leaf in an apple seedling rootstock nursery. Predaceous mites (all stages) increased to 0.3-0.4 per leaf after releases and predator prey ratios of < or = 1:3-7 provided pest regulation thereafter. Such low-density releases were thought to be effective because multiple dispersal bouts allowed predators to locate widely distributed spider mites (on 2-6% of leaves). A random-diffusion model simulating predator dispersal (incorporating wind speed and direction parameters) adequately explained movement and pest control patterns. An upright, dense, uniform planting of apple seedlings was an effective producer and recipient for dispersing predators and these attributes seemed to explain why biological control was so effective. Low-density releases of N. fallacis for control of T. urticae are predicted to be less effective on other crops with less prominent profiles and soil coverage.

First Report of the Pathogenicity of Colletotrichum gloeosporioides on Invasive Ferns, Lygodium microphyllum and L. japonicum, in Florida.

Lygodium microphyllum (Cav.) R.Br. (Old World climbing fern) and L. japonicum (Thunb.) Sw. (Japanese climbing fern), in the family Schizaeaceae, are among the most invasive weeds in Florida (1). L. microphyllum invades fresh water and moist habitats in south Florida, while L. japonicum has spread in relatively well-drained habitats from Texas to North Carolina and central Florida. Some potted plants of both Lygodium spp. grown in shadehouse as well as in full sunlight developed discolored spots on pinnules (foliage), which coalesced and resulted in browning and dieback of severely infected vines. Symptomatic foliage obtained from these plants was surface-sterilized by immersing in a 15% solution of commercial bleach for 90 s, followed by a series of four rinses with sterile deionized distilled water. Disks (4 mm in diameter) of pinnules were cut from the junction of discolored and healthy tissues and placed on potato dextrose agar (PDA). A fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. was consistently isolated from these disks. Fungal colonies produced abundant conidia on PDA. Conidia were hyaline, straight, cylindrical, averaging 14.7 µm (range 12.5 to 17.5 µm) × 5.0 µm (range 3.8 to 7.5 µm), and similar to those described for C. gloeosporioides (2). To confirm the pathogenicity of C. gloeosporioides on L. microphyllum and L. japonicum, Koch's postulates were performed. A fungal isolate was grown on PDA for 3 weeks, after which 10 ml of sterile deionized distilled water was added to the culture and agitated to dislodge conidia. The conidial suspension was strained through three layers of cheesecloth to remove hyphal fragments, and its concentration was adjusted to 1.7 × 106 conidia/ml. Foliage of healthy L. microphyllum and L. japonicum plants grown in 500-ml containers was sprayed with the conidial suspension until runoff. Plants were covered with plastic bags whose inner sides were misted with water to maintain high humidity and placed in a growth chamber under 12 h of fluorescent light per day. Temperature and relative humidity in the chamber ranged from 26 to 29°C and 44 to 73%, respectively. Plastic bags were removed after 3 days, and plants were further incubated for 3 weeks in the same growth chamber. Control plants were sprayed with sterile water, covered with plastic bags, and exposed to the same temperature, light, and humidity regime as those of the fungus-inoculated plants. Small, discolored foliar spots appeared 3 days after fungus inoculation. These spots were similar to those observed on pinnules of potted plants that originated from shadehouse and outdoor environments. Within 3 weeks after inoculation, the foliage of L. japonicum developed abundant discolored spots that led to edge browning and wilting of the pinnules. L. microphyllum had similar but more severe symptoms, with plants suffering as much as 50% dieback. C. gloeosporioides was consistently reisolated from the symptomatic tissues of both fern species. No symptoms appeared on the water-inoculated plants. To our knowledge, this is the first record of C. gloeosporioides pathogenicity on L. microphyllum and L. japonicum. References: (1) R. W. Pemberton and A. P. Ferriter. Am. Fern J. 88:165, 1998. (2) B. C. Sutton. Colletotrichum: Biology, Pathology and Control. CAB International, Wallingford, Oxon, UK, 1992.

First Report of the Pathogenicity of Rhizoctonia solani on Salvinia molesta and S. minima in Florida.

Salvinia molesta Mitchell (giant salvinia) and S. minima Baker (common salvinia) are exotic aquatic ferns that have invaded drainage basins in Texas, Louisiana, Alabama, Arizona, California, Florida, Georgia, Hawaii, Mississippi, North Carolina, and Oklahoma (2). These ferns rapidly colonize bodies of water and form thick mats, displace native species, disrupt recreational activities like boating and fishing, block drainage and irrigation intakes, interfere with electricity generation, and degrade water quality (1). Patches of water-soaked lesions were observed on the pinnules and rachises of screenhouse-grown S. molesta plants in Florida. Mycelia spread centrifugally from these patches and caused diseased plants to disintegrate and sink. Brown-to-black sclerotia were formed on and around the disintegrated plants. A fungus was consistently isolated from symptomatic tissues of S. molesta plants. Seven-day-old cultures turned buff-colored and produced sclerotia on potato dextrose agar, while cultures on water agar were hyaline and produced black sclerotia. Both types of sclerotia were not differentiated into rind and medulla. The mycelia branched at right angles from the main hyphae, were constricted at the base of the angle, and had a septum after the constriction. Vegetative cells were multinucleate. The fungus was identified as Rhizoctonia solani Kühn (3,4). Koch's postulates were performed to confirm pathogenicity on S. molesta and S. minima. Seven-day-old cultures of R. solani that were grown in potato dextrose broth were filtered through four layers of cheesecloth and washed with distilled water. Fourteen grams of the mycelial residue was suspended in 28 ml of distilled water and macerated in a small blender for 30 s to obtain a mycelial suspension. Healthy S. molesta and S. minima plants grown in screenhouse-tanks were immersed in tap water supplemented with 1 drop per 4 liters of surfactant (Tween 80), rinsed thoroughly, and approximately 40 g of the plants was floated in plastic jars (18.5 cm diameter × 7.5 cm high) filled to a depth of 5 cm with tap water. Three jars each of S. molesta and S. minima were misted with 1.5 ml of the mycelial suspension. Individual jars were covered with a clear plastic lid with a 2.5-cm-diameter hole in the center for ventilation. These jars were placed in a growth chamber maintained at 28 (+1)°C and 12-h fluorescent light cycles. Typical water-soaked lesions appeared on pinnules within 3 to 7 days, spread rapidly, and resulted in disintegration of pinnules and rachises. R. solani was consistently reisolated from symptomatic tissues of both Salvinia species. To our knowledge, this is the first report confirming pathogenicity of R. solani on S. molesta and S. minima. This fungus should be further evaluated as a potential mycoherbicide for control of Salvinia species. References: (1) K. L. S. Harley and D. S. Mitchell. J. Aust. Inst. Agric. Sci. 47:67, 1981. (2) C. C. Jacono et al. Castanea 66:214, 2001. (3) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN, 1991. (4) C. C. Tu and J. W. Kimbrough. Bot. Gaz. 139:454, 1978.

Plant-related factors influence the effectiveness of Neoseiulus fallacis (Acari: Phytoseiidae), a biological control agent of spider mites on landscape ornamental plants.

The predatory mite Neoseiulus fallacis (Garman) was evaluated as a biological control agent of herbivorous mites on outdoor-grown ornamental landscape plants. To elucidate factors that may affect predator efficiency, replicated tests were conducted on 30 ornamental plant cultivars that varied in relationship to their generalized morphology (e.g., conifers, shade trees, evergreen shrubs, deciduous shrubs, and herbaceous perennials), production method (potted or field grown), canopy density, and the prey species present on each. Plant morphological grouping and foliar density appeared to be the most influential factors in predicting successful biological control. Among plant morphological groups, N. fallacis was most effective on shrubs and herbaceous perennials and less effective on conifers and shade trees. N. fallacis was equally effective at controlling spider mites on containerized (potted) and field grown plants, and there was no difference in control of mites on plants with Tetranychus spp. versus those with Oligonychus or Schizotetranychus spp. Moderate to unsuccessful control of spider mites by N. fallacis occurred mostly on tall, vertical plants with sparse canopies. Acceptable spider mite control occurred in four large-scale releases of N. fallacis into production plantings of Abies procera, Thuja occidentalis 'Emerald', Malus rootstock, and Viburnum plicatum 'Newport'. These data suggest that N. fallacis can be an effective biological control agent of multiple spider mite species in a range of low-growing and selected higher growing ornamental plants.

First U.S. Report of Pseudocercospora paederiae Leaf Spot on the Invasive Exotic Paederia foetida.

Paederia foetida L., commonly referred to as skunk vine, is a native of eastern and southern Asia and was introduced into the United States prior to 1897. By 1916 it was already a troublesome weed in central Florida. It is a fast growing perennial twining vine (up to 7 m) with a woody rootstock adapted to a wide range of light, soil, water, and salt conditions (4). Naturalized in Florida, Georgia, Hawaii, Louisiana, North Carolina, South Carolina, and Texas, it occurs most often in disturbed areas. In Florida, where it is listed by the Florida Department of Agriculture and Consumer Services as a noxious weed, it invades various native plant communities including sandhills, flood plains, and upland mixed forests, where it creates dense canopies leading to injury or death of native vegetation and structural alteration of the native plant community (2,4). Current work underway to find biological control agents for invasive weeds led to the discovery in central Florida of a skunk vine plant with irregular to angular, sunken leaf spots ranging in color from shiny black to dark brown, some with tan centers and dark brown borders. Leaf spots had coalesced in some areas, blighting portions of leaves. Pseudocercospora paederiae (Sawada ex) Goh & Hsieh (1,3) was recovered from these leaf spots. Fruiting was amphigenous (chiefly epiphyllous) with globular or subglobular stromata, formed singly or coalesced, 37.2 µm wide (range = 19.9 to 62.3 µm). Conidia were hyaline to faintly olivaceous, with up to 6 septa, straight to mildly curved, measuring 49.6 µm (range = 18.8 to 72.3 µm) × 4 µm (range = 3 to 5 µm). To confirm Koch's postulates, a healthy, vigorous P. foetida plant in a 12 liter pot was spray-inoculated with 47 ml of a conidial suspension (13,000/ml) of P. paederiae. The plant was covered with a clear plastic bag to create a moist atmosphere and kept at room temperature (25°C) for 3 days after which it was uncovered and moved into a greenhouse. The greenhouse temperature fluctuated between 15°C (nighttime) and 29°C (daytime). Symptoms started appearing after 2 weeks, becoming more prominent by the third and fourth week. The inoculated plant showed irregular to angular dark brown to black leaf spots with dark brown borders. Necrosis along veins was observed and severely infected leaves abscised. The fungus was consistently recovered from inoculated symptomatic leaf tissue. Continued incubation of the plant under greenhouse and outdoor raised bench conditions eventually resulted in the secondary infection and leaf spotting of new foliage. P. paederiae was recovered from these secondary lesions. P. paederiae has been previously reported from Taiwan, China, and Japan. This represents the first report of the pathogen in the Western Hemisphere. Pathogenicity tests suggest possible application as a mycoherbicide. References: (1) C. Chupp. 1953. A Monograph of the Fungus Genus Cercospora. Cornell University Press. Ithaca, New York. (2) G. Gann and D. Gordon. Natural Areas J. 18:169, 1998. (3) W. H. Hsieh and T. K. Goh. 1990. Cercospora and Similar Fungi from Taiwan. Maw Chang Book, Taiwan, Republic of China. (4) K. A. Langeland and K. C. Burks, eds. 1998. Identification & Biology of Non-Native Plants in Florida's Natural Areas. University of Florida Press, Gainesville, FL.