Spores of this fungus are contained in a gelatinous matrix and may be moved by insects, birds, or contaminated tools.

Method	Instructions	NAPIS Survey Method
Visual	Use visual survey to collect symptomatic plant material.	3031 - General Visual Observation

It may be useful to visually survey host species that are known to be particularly susceptible to this fungus, such as Buxus semperviens "Suffruticosa", which is hardy to USDA Zone 5. Surveys should occur wherever host plants are abundant. As the hosts are ornamentals, surveys could occur in large scale container and field nurseries, gardens, parks, or residential areas. As this fungus may move through international trade (through ornamental plants for planting), nurseries are a logical survey site. It is unknown whether other members of the family Buxaceae, such as Pachysandra), are also susceptible so any suspicious samples from these plants should be collected as well. Submit adequate amounts of suspect leaf and stem material when possible. This helps ensure that there is sufficient material if downstream diagnostic techniques are required. Refrigerate samples while awaiting shipment to the diagnostic laboratory. Place leaves without paper towel in a sealed and labeled ziplock bag. Disease spreads rapidly throughout infected plants when conditions are warm and humid, and in shady areas. Most boxwood cuttings are initially rooted and propagated in humidity chambers or tents, and young boxwood liners are purposely grown under shady conditions. These production conditions are also conducive to the pathogen; therefore young boxwoods are more at risk to infection.

Sporulation of the pathogen can be seen on the underside of the infected leaves. Under high humidity, white fuzzy spore masses (called sporodochia) containing large numbers of conidia are sometimes visible to the naked eye or with a hand-lens on infected stem and leaf tissue.

Dark or light brown spots on leaves with dark borders (often in a circular pattern), black streaks on stems, straw to bronze colored blighted foliage, and defoliation are observed. In some cases, blighting and defoliation can occur suddenly with complete leaf loss in severe cases.

Infected leaves can be aggregated in expanded patches. Infected leaf tissue will eventually expand to the petiole and shoot of the leaf. Leaf spots can coalesce, covering the entire leaf surface. Infected shoots can have multiple dark brown or black lesions, either linear or diamond in shape. The black streaks found on stems progress from the bottom of the plant up. New young shoots continue to develop on healthy twigs.

The fungus has not been recovered on the root system and often the root systems remain healthy and intact.

ID/Diagnostic: Multiple

Morphological: Cylindrocladium spp. are morphologically differentiated mainly by the shape of the vesicle and characteristics of the conidia but misidentifications can occur, mainly due to cultural conditions that may influence these characters.

If a state has not yet detected Cylindrocladium pseudonaviculatum , any symptomatic suspect plant survey samples taken, whether by PPQ or the state, need to go through screening at their NPDN and/or state lab and examined by a plant pathologist/diagnostician. The attached pest alert was distributed to all NPDN labs which should include state departments of agriculture with their own laboratories, so they should already be aware of the issue. For states doing surveys that do not have a state or university plant clinic or plant pathology lab to screen the samples, please contact Phil Mason in the PPQ Western Region Office or Leon Bunce in the Eastern Region PPQ Office who will coordinate with Joel Floyd to see where plant samples can be sent for screening by a diagnostician.

This is a morphological fungal identification (molecular is not necessary or required) and new state records are confirmed by our national mycologists. See the pest alert attached for guidance and the address for sending samples screened as described above to the national mycologist. After the first state record is confirmed by them, any subsequent confirmations can be handled by the state, unless they are not sure of an identification, or require a national confirmation for their purposes. Those state or university labs confirming their own new positives should keep the SPRO and SPHD informed of all the localities that are positive. Until a we know about a potential regulatory stance decided for this pathogen, states should also retain vouchers for each positive locality, i.e., a culture or semi-permanent microscope slide with diagnostic structures, in case they may be needed for national confirmation if required by a state.

This species was initially misidentified as Cylindrocladium scoparium in the United Kingdom (Henricot and Culham, 2002). Cy. scoparium is the most misidentified species in the genus most likely due to variability in morphological characteristics used in identification. Cy. pseudonaviculatum can be differentiated from Cy. scoparium as it has "one-septate conidia and ellipsoidal vesicles with pointed or papillate apices"; Cy. scoparium tend toward globiose to ob-pyroid vecicles and lacks the pointed, lanceolate vesicles (Henricot and Culham, 2002). Cy. pseudonaviculatum can occur with Volutella buxi (teleomorph Pseudonectria rousseliana) that causes Volutella blight. V. buxi produces pink to orange spore masses on infected tissues, and under the microscope it is fairly easy to distinguish these fungi. Care must be taken to rule out the presence of Cy. pseudonaviculatum when Volutella is observed, as both pathogens may be present in the tissue. Other pathogens that affect Buxus spp. in the United States are Phytophthora parasitica, Macrophoma candolleri (Macrophoma leaf spot), and nematodes. Phytophthora parasitica can be found on all cultivars of B. sempervirens (American boxwood) and causes wilting and discoloring of the foliage. Macrophoma candolleri is considered a secondary invader and causes raised tiny black spots on the underside of leaves. Nematodes cause wilting, stunting, and yellowing of the foliage. All of these pathogens, as well as Paecilomyces buxi (=Verticillium buxi), may cause Boxwood decline. This complex is poorly understood, but can lead to poor plant growth, small leaves, and defoliation or dieback.

In order to correctly identify Cy. pseudonaviculatum as a new species, Henricot and Culham (2002) used a variety of methods including morphological characteristics, sequencing the ribosomal 5.8S RNA gene and the flanking internal transcribed spacers (ITS), the β-tubulin gene, and the high mobility group (HGM) of the MAT2 mating type gene. Isolation: Henricot and Culham (2002) incubated diseased leaf and stem pieces of Buxus spp. in damp chambers at 20°C (68°F) to induce sporulation. Isolates were single spored and subcultured weekly on potato carrot agar (PCA) (carrot agar 20g/L, potato agar 20g/L, ampicillin 30 mg/L, streptomycin 133 mg/L) (Henricot and Culham, 2002). To determine morphology "Single spores of 14 isolates were plated onto carnation-leaf agar (CLA) and incubated at 25°C [77°F] under near-ultraviolet light. The plates were examined after 7 d or until sporulation occurred (no later than 9 d) and only conidiophores on the carnation leaves were examined. For each isolate, mounts were prepared in lactic acid with aniline blue (0.2 g/100 mL), and measurements of at least 30 conidia, vesicles, stipes, branches and phialides were made at 1000x magnification with an optical microscope (Henricot and Culham, 2002).

Recently found in North Carolina, Connecticut, and Virginia.