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 Bipolaris sacchari Causing Leaf Spot on Lygodium japonicum and L. microphyllum in Florida.

Lygodium japonicum (Thunb.) Sw. (Japanese climbing fern) and L. microphyllum (Cav.) R.Br. (Old World climbing fern) are invasive, noxious weeds in Florida. During 2001, L. japonicum sporelings were collected from natural sites in Hamilton, Highland, and Madison counties and transported to Broward County (Fort Lauderdale) for research use. During February 2002, leaf spots were observed on pinnules (leaflets) of these containerized plants growing in a shadehouse. A fungus with Bipolaris-like spores was isolated from affected pinnules and purified and stored for future evaluation. In early 2005, the fungus was grown on 1.5% water agar, with sterile, wheat straw pieces embedded in the agar surface, at 26°C and a 12-h photoperiod using cool-white fluorescent and Verilum full-spectrum bulbs. Conidia were 80.5 ± 14.5 µm (range 53.2 to 123.4 µm) × 15.1 ± 1.6 µm (range 12.1 to 19.4 µm), pale brown, slightly curved, narrowly ellipsoid, without a protuberant hilum, distoseptate (8 ± 1, range 6 to 10), and germinated from both polar cells. Conidiophores were septate and smooth. Conidiogenous nodes were smooth. On the basis of these characteristics, the fungus was identified as Bipolaris sacchari (E. Butler) Shoem. (1,2). Pathogenicity toward L. japonicum and L. microphyllum was determined using conidia produced on potato dextrose agar at 26°C and a 12-h photoperiod. A 1 × 106 conidia ml-1 suspension was sprayed until runoff on healthy plants grown in 450-ml containers. Control plants were sprayed with sterile water. There were four replicate plants per treatment. Plants were covered with plastic bags to maintain high humidity and placed in a growth chamber with a 12-h photoperiod at 28°C (light cycle) and 22°C (dark cycle). Bags were removed after 72 h, and small (1 to 2 mm), water-soaked spots were evident throughout the plant canopy on both Lygodium spp. Plants were incubated for three more weeks under the same photoperiod and temperatures with ˜70% relative humidity (light cycle) and ˜45% relative humidity (dark cycle), and then evaluated for disease. At least 50% of L. microphyllum pinnules and 25% of L. japonicum pinnules on each inoculated plant had small (1 to 2 mm), brown leaf spots or larger (approximately 5 mm) necrotic spots. B. sacchari was reisolated from both types of spots from both Lygodium spp.; there was no evidence of fungal sporulation on the plants. No symptoms were apparent on control plants. To our knowledge, this is the first report of B. sacchari on a non-Poaceae host in Florida. References: (1) J. L. Alcorn. Ann. Rev. Phytopathol. 26:37, 1988. (2) A. Sivanesan. Graminicolous Species of Bipolaris, Curvularia, Dreschslera, Exserohilum and Their Teleomorphs. CAB International, Wallingford, Oxon, UK, 1987.

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.

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.