First Report of White Pine Blister Rust on Rocky Mountain Bristlecone Pine.

White pine blister rust caused by Cronartium ribicola was introduced into North America in the early 20th century and is spreading throughout the range of five-needle pines. In northern Colorado, this pathogen was first observed in 1998 on limber pine (Pinus flexilis) (1). It has not been reported on Rocky Mountain or Great Basin bristlecone pine (Pinus aristata and P. longaeva, respectively) in nature. However, Rocky Mountain bristlecone pine is susceptible to the disease when artificially inoculated (2). In October 2003, a Rocky Mountain bristlecone pine was found infected with C. ribicola in the Great Sand Dunes National Monument, Alamosa County, Colorado. Seven branch cankers were observed on the tree. Cankers ranged in length from 15 to 41 cm and were estimated to be approximately 5 to 7 years old. Distinct C. ribicola branch symptoms were observed, including flagging, spindle-shaped swellings, and 6 mm long aecial scars. A branch was deposited at the Colorado State Herbarium. Microscopic examination of spores within remnant aecial blisters revealed aeciospores characteristic of C. ribicola (yellow-orange, ellipsoid, verrucose, and 19 × 25 µm). Cankers were only observed on one bristlecone pine. However, most limber pines in the area were infected with C. ribicola, including a limber pine less than 1 m from the infected bristlecone pine. To our knowledge, this is the first report that shows Rocky Mountain bristlecone pine can become infected naturally, and the pathogen is further south in Colorado on limber pine than previously reported. These observations suggest the need for a more complete investigation of this disease on bristlecone pines. References: (1) D. W. Johnson and W. R. Jacobi. Plant Dis. 84:595, 2000. (2) B. R. Stephan, Allg. Forst Z. 28:695, 1985.

Identification of Fungi and Fungal Pathogens Associated with Hypolixus haerens and Decayed and Cankered Stems of Amaranthus hybridus.

Discoloration, cankers, and decay in branches, stems, and root collars of Amaranthus hybridus were observed in Bloemfontein, South Africa. Examination of symptomatic stems revealed larval galleries of the pigweed weevil (Hypolixus haerens). The objectives of this study were to: identify the most common fungal species associated with this damage, determine if the adult pigweed weevil might be a vector for the fungi, and test if the associated fungi can cause the stem canker disease observed in the field. The most common fungal species isolated were Fusarium subglutinans from discolored tissues adjacent to insect galleries (42%), F. subglutinans from weevil larvae (29%), the Alternaria tenuissima group from adult weevils (31%), and the A. tenuissima group from cankered stems (40%). Three of the seven most common fungal species produced cankers following wounding and inoculation, with F. sambucinum and F. oxysporum being the most aggressive. Although fungal species compositions differed (P < 0.01) among the four tissue/insect stage combinations tested, all four had the same major fungal species, suggesting the pigweed weevil as a vector for the Fusarium pathogens. There is significant potential for yield loss affiliated with this insect-fungal association. The identification of this insect-fungal relationship and the pathogens involved in disease set the stage for further research on the etiology and disease management of this important insect-fungal relationship.

Infection, Colonization, and Disease of Amaranthus hybridus Leaves by the Alternaria tenuissima Group.

With the increased use of Amaranthus hybridus as a leafy-vegetable crop in Africa and the recent identification of Alternaria leaf spot on this host in southern Africa, the role of this potentially damaging pathogen was investigated. The goals of this study were to test the pathogenicity of the Alternaria tenuissima group, determine how these fungi infect Amaranthus hybridus leaves, and examine the colonization pattern within host tissues. Asymptomatic leaves of Amaranthus hybridus were collected from two field sites in South Africa. Eight A. tenuissima group isolates collected from these leaves were used in inoculation experiments conducted in both greenhouse and growth chamber studies. Scanning electron microscopy revealed A. tenuissima-like conidia germinating on leaf surfaces and mycelia entering leaves only through stomata of both field-collected and artificially inoculated leaves. Unwounded, inoculated leaves had no symptoms, and light-microscopy observations of both asymptomatic field-collected and unwounded and inoculated leaves revealed hyphae in mesophyll tissue growing intercellularly with no host cell penetration or host-cell response. Seven of the eight isolates produced brown to black, circular to oval, necrotic lesions only at the wound site of injured and inoculated leaves. These results confirm that isolates of the A. tenuissima group can infect and colonize Amaranthus hybridus leaves in a manner consistent with other endophytic fungi, and suggest that these fungi can act as latent leaf pathogens when the host is altered by wounding.

Water stress and Sphaeropsissapinea as a latent pathogen of red pine seedlings.

• The role of water stress in the initiation of collar rot by Sphaeropsis sapinea in asymptomatically colonized Pinus resinosa seedlings is reported. • Mortality and frequency of identification of the pathogen was quantified for seedlings subjected to different water regimes or watering regime-fungicide (benomyl) combinations in glasshouse experiments. • In experiment 1, seedling mortality ranged from 8% of repeatedly watered seedlings to 50% of those in the driest regime; data analysis indicated a high probability that mortality was not independent of watering regime. Seedlings developed symptoms resembling those of Sphaeropsis collar rot, and S. sapinea was identified from living (42%) and dead (92%) seedlings. In experiment 2, mortality of repeatedly watered seedlings was low, irrespective of fungicide application. For nonwatered seedlings, however, mortality was greater among seedlings not treated with fungicide (61%) than among benomyl-treated seedlings (37%); data analysis indicated a high probability that mortality was not independent of fungicide treatment. • Sphaeropsis sapinea can act as a latent pathogen; physiological alteration, through water stress, can effect release from the quiescent condition to result in rapid disease development.

Differences in Aggressiveness of Sphaeropsis sapinea RAPD Marker Group Isolates on Several Conifers.

Seedlings of Scot's pine varieties East Anglia and Austrian Hills, red pine, mugho pine variety Pumileo, Colorado blue spruce, Douglas-fir, and balsam fir were wounded and inoculated with water agar plugs colonized by isolates of the two random amplified polymorphic DNA (RAPD) marker groups (A and B) of Sphaeropsis sapinea. Isolates were obtained from hosts in Michi-gan, Minnesota, and Wisconsin. Symptom severity (distance from the inoculation site at which necrotic needles were present) resulting from inoculations with each group A isolate exceeded that from inoculations with each group B isolate on all hosts except Colorado blue spruce. Hosts varied considerably in their responses to group A isolates. Based on symptom severity, East Anglia Scot's pine was most susceptible and balsam fir was least susceptible when inoculated with group A isolates. The pathogen was recovered from both symptomatic and asymptomatic seedlings inoculated with isolates of either group. Results emphasize the importance of characterizing a RAPD marker group(s) of S. sapinea encountered in the field or used in research; the need for comparative evaluations of resistance among coniferous genera, species, and varieties to S. sapinea of both groups; and the potential for asymptomatic persistence of S. sapinea from both groups in or on several coniferous hosts.

First Report of Alternaria tenuissima as a Leaf Pathogen of Amaranthus hybridus.

Amaranthus hybridus is an important alternative leafy-vegetable crop with the potential for increased commercial production in southern Africa and other semi-arid regions of the world (2). In May 1998, extensive leaf spotting was observed on A. hybridus at Potchefstroom, South Africa. Many of the leaves had symptoms and most were severe. Symptoms were dark brown to black, circular to oval, necrotic lesions with a diameter ranging from <1 mm up to 7 mm. Larger lesions had tan centers. Tissues adjacent to the leaf spots remained green. Alternaria tenuissima was isolated from 43% of 40 lesions sampled making up 89% of the isolates recovered. A. tenuissima was isolated from asymptomatic leaves of 5-month-old A. hybridus plants sampled from the same site in April 1997 (1). The foliar symptoms observed on A. hybridus in Potchefstroom were reproduced by inoculating wounded leaves of A. hybridus with single-spore A. tenuissima isolates obtained from asymptomatic leaves collected at Potchefstroom. Eight isolates were selected for pathogenicity tests conducted in a growth chamber. A. hybridus leaves grown from seed in a greenhouse were placed in moist chambers and wounded with a needle (0.5 mm) at leaf center; one 5-mm-diameter, colonized potato dextrose agar plug of each of the isolates was placed on the center of each leaf. A sterile plug was used as a control. Moist chambers were placed in a growth chamber set at 25°C day and 20°C night and provided artificial light for 16 h per day. In a greenhouse (average temperature 25°C day, 17°C night), a conidial suspension (105 conidia per ml of sterile, distilled water) was applied to an individual leaf of each of 14 plants with an atomizer. Sterile, distilled water was applied to control leaves. Leaves were then wounded with a needle (0.5 mm) at leaf center. Treatments were assigned randomly and the experiments were repeated. Symptoms were first observed at 14 and 18 days (growth chamber and greenhouse, respectively). Seven of the eight isolates caused necrotic lesions with an average diameter of 3 mm (both growth chamber and greenhouse). Symptoms were observed on an average of 56 and 82% of the inoculated leaves (growth chamber and greenhouse, respectively). The range of symptoms was the same as that observed in the field, but symptoms were only observed at the wounds. Controls remained green and showed no symptoms. A. tenuissima was recovered from necrotic lesions of surface-disinfested, inoculated leaves (average 93 and 68%; growth chamber and greenhouse, respectively), never recovered from growth chamber controls, and seldom recovered from greenhouse controls (5%). These results suggest that A. tenuissima is a leaf-spot pathogen of A. hybridus and wounding might trigger disease expression. A minimal amount of leaf spotting of this leafy-vegetable crop can cause total crop loss. References: (1) J. T. Blodgett et al. S. Afr. J. Sci. 94:xviii, 1998. (2) W. J. Swart et al. S. Afr. J. Sci. 93:xxii, 1997.

First Report of Fusarium sambucinum, F. oxysporum, and F. subglutinans Associated with Stem Decay of Amaranthus hybridus in South Africa.

Amaranthus hybridus (common name: amaranth) is a fast-growing crop with nutritious leaves and seeds that is cultivated in semi-arid regions throughout the world. In South Africa, cultivation of this crop as a leafy vegetable is increasing. In autumn 1997, extensive tissue discoloration and decay were observed in branches, stems, and root collars of mature A. hybridus in Bloemfontein, Free State Province. Symptoms included discolored phloem, xylem, and pith, black cankers, and weakened stems prone to wind breakage. Examination of these tissues revealed larval galleries of the pigweed weevil (Hypolixus haerens), the main insect pest of A. hybridus in South Africa (1). Six-month-old A. hybridus stems were split and small samples of discolored tissue adjacent to the larval galleries of each stem and the associated larvae were placed aseptically on corn-meal agar containing streptomycin and incubated for 4 to 7 days. The seven fungi most frequently isolated from discolored stem tissues (n = 166) were Fusarium subglutinans (46%), a Phomopsis sp. (11%), Alternaria alternata (10%), F. oxysporum (9%), F. solani (5%), a Phoma sp. (5%), and F. sambucinum (4%). The nine fungi most frequently isolated from larvae (n = 90) were F. subglutinans (46%), F. solani (8%), F. equiseti (8%), F. oxysporum (7%), A. alternata (6%), a Phomopsis sp. (4%), F. proliferatum (3%), F. sambucinum (2%), and a Phoma sp. (2%). Stems of greenhouse-grown A. hybridus were inoculated with the seven most common species isolated from the discolored stem tissues. One isolate of each species was used. Inoculations involved wounding stems by removing approximately 36 mm2 of the epidermis 5 cm above the soil, placing a colonized water agar plug on the wound, and wrapping Parafilm around the stems at the wound site. Wounded and nonwounded (untreated) controls were also included. A noncolonized water agar plug was applied to wounded controls but not to nonwounded controls. Ten plants per isolate and 10 wounded and nonwounded control plants were used in each of two separate trials (180 total plants). Treatments were assigned randomly. Four weeks after inoculation, canker lengths were measured and stem sections were surface disinfected and transferred to water agar plates. The presence of the fungi was confirmed after 20 days. Only F. sambucinum, F. oxysporum, and F. subglutinans caused cankers with frequencies of 100, 100, and 65% (n = 20), and mean lesion lengths of 30, 26, and 10 mm, respectively. Lesions were never observed on either of the controls. Discoloration and cankers were similar to that observed in the field. F. sambucinum, F. oxysporum, and F. subglutinans were recovered from 65, 50, and 60% of the tissues, respectively, and none of the Fusarium spp. were recovered from the control treatments (n = 20 for all). In artificial inoculations, these species can act as pathogens independent of the pigweed weevil and are likely the cause of the discoloration, decay, and cankers observed in branches, stems, and root collars of mature A. hybridus. However, there are no prior reports of a Fusarium sp. causing disease on A. hybridus, and H. haerens larvae were observed in all symptomatic stems in the field. Further studies are needed to determine the potential for significant disease loss associated with this insect-fungal association and the potential role of these fungi in further weakening Amaranthus stems that are colonized by H. haerens. Reference: (1) S. vdM. Louw et al. Afr. Crop Sci. J. 3:93, 1995.

First Report of Botryosphaeria dothidea Basal Canker of Pistachio Trees in South Africa.

Pistachio (Pistacia vera) cultivation is a relatively new industry in South Africa with tremendous economic potential. However, disease problems could develop in time. The pathogens of pistachio found in other parts of the world plausibly will spread to South Africa and new pathogens never recorded on this host may develop here. In the summer of 1998, 2-year-old rootstocks of P. atlantica and P. integerrima displaying basal cankers and discolored phloem and xylem were observed in the Prieska district of the Northern Cape province. The objective of this study was to identify the causal agent(s). Cankered stems were split and small samples of discolored wood were aseptically placed on corn-meal agar containing streptomycin and incubated for 5 days. Fungi that grew into the agar were transferred to 1.5% water agar dishes containing pine needles to aid sporulation. Species isolated from stems included Fusarium spp. (44%), Fusicoccum aesculi (anamorph of Botryosphaeria dothidea) (23%), a Cytospora sp. (19%), a Chaetomium sp. (3%), and several nonsporulating mycelial fungi (11%). Inoculum of suspected pathogens (F. aesculi, the Cytospora sp., and two Fusarium spp.) were prepared by culturing fungi in petri dishes on potato dextrose agar overlaid with sterile cheesecloth strips (approximately 15 × 25 mm) until the strips were completely colonized. Greenhouse inoculations involved wounding P. atlantica stems by removing the bark (approximately 3 × 6 mm) approximately 10 cm above the soil, and wrapping the colonized cheesecloth followed by Parafilm around the stems at the wound site. Eight plants per isolate and eight wounded and nonwounded (untreated) control plants were used. Noncolonized cheesecloth was applied to wounded controls but not to nonwounded controls. Treatments were assigned randomly. Eight weeks after inoculation, the surrounding bark was removed from all treated shoots and the cambium was examined for discoloration. The length of each cambial lesion was measured and stem sections were surface disinfested and transferred to 1.5% water agar dishes, and the presence of the inoculated fungi was confirmed. Only F. aesculi was pathogenic to P. atlantica and produced cankers on all stems. The fungus was recovered from all tissues sampled 3 cm beyond the wound sites, 88% of those sampled from the wound sites, and none of the control treatments. Discoloration of the phloem and xylem was similar to that observed in the field. The mean canker length was 41 mm on trees with a mean stem diameter of 7 mm. The appearance of B. dothidea, in addition to B. obtusa (1), on pistachio justifies the need for establishing a disease management program for pistachio in South Africa. Reference: (1) W. J. Swart and W.-M. Botes. Plant Dis. 79:1036, 1995.

Monoterpene and Phenolic Compound Concentrations in Water-Stressed Red Pine Inoculated with Sphaeropsis sapinea.

ABSTRACT Changes in monoterpene and phenolic compounds resulting from water stress and colonization by Sphaeropsis sapinea were examined for 9- and 11-year-old red pine trees in a plantation and 3-year-old seedlings in a growth chamber. Four treatments were assigned at random to individual trees in the field: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. In the growth chamber, seedlings were either not watered (water stressed) or watered daily (nonstressed). Shoots were inoculated with agar plugs colonized with either S. sapinea isolates of morphotype A and B (field) or only isolates of morphotype A (growth chamber). Nine monoterpenes were detected in tissue extracts; the most common were alpha-pinene (59 to 74% of the total), beta-pinene (13 to 33% of the total), and delta-3-carene (1 to 5% of the total). Shoots inoculated with isolates of morphotype A had more severe symptoms and produced higher concentrations of monoterpenes in both experiments compared with the controls. In the growth chamber, inoculations with isolates of morphotype A caused higher concentrations of phenolics compared with the controls. In the field experiment, monoterpenes increased in quantity only in shoots of stressed trees inoculated with isolates of morphotype A. Isolates of morphotype B caused few symptoms and did not alter monoterpene concentrations. Increases in monoterpenes do not appear to be involved in the response to infection by morphotype A in nonstressed trees, and the role of phenolics is unclear. However, these results are consistent with previous observations that monoterpenes may be involved in the differences in aggressiveness between morphotypes on red pine.

Differential Inhibition of Sphaeropsis sapinea Morphotypes by a Phenolic Compound and Several Monoterpenes of Red Pine.

ABSTRACT The in vitro effects of a red pine phenolic compound (pinosylvin), a phenolic compound common to other species (tannic acid), and the major red pine monoterpenes (alpha-pinene, beta-pinene, and delta-3-carene) on spore germination and mycelial growth of Sphaeropsis sapinea were examined. Two A and two B morphotype isolates were used. At 88 mug/mm(2), pinosylvin inhibited spore germination of all four isolates (98 to 100%). At 8.8mug/mm(2), spore germination of B isolates was inhibited more than that of A isolates (73 versus 30%). Pinosylvin also inhibited mycelial growth of B isolates more than that of A isolates (84 versus 13% at 88 mug/mm(2)). Tannic acid stimulated or had little affect on spore germination and had little affect on mycelial growth of either morphotype. Spore germination of B isolates was inhibited more than that of A isolates by beta-pinene at saturation (79 versus 37%). Spore germination of B isolates was inhibited and germination of A isolates was stimulated by delta-3-carene below saturation (49 versus -7%). Mycelial growth of B isolates was inhibited more than that of A isolates by all monoterpenes at saturation. Differences observed between morphotypes below saturation were significant only for beta-pinene. These results demonstrate the biological activity of a phenolic compound and monoterpenes that occur in red pine. The differential responses might provide means of distinguishing morphotypes and offer a potential explanation for ecological specialization.

Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine.

ABSTRACT The aggressiveness of Sphaeropsis sapinea isolates was compared on water-stressed and nonstressed 3-year-old red pines (Pinus resinosa) in greenhouse and growth chamber experiments. Water was withheld from stressed seedlings to achieve mean predawn needle water potentials (psi(PD)) above -1.9 MPa. The lowest mean psi(PD) of well-watered seedlings was maintained at or above -0.8 MPa. Young shoots were inoculated by placing colonized agar plugs on wounds made by removing a needle fascicle. Two isolates of each recognized morphotype (A and B) were used in the greenhouse experiment and two isolates of morphotype A were used in the growth chamber experiment. After 4 weeks, isolates of morphotype A caused more severe symptoms and could be recovered farther from the inoculation site on water-stressed than on nonstressed trees in both experiments. In the greenhouse experiment, isolates of mor-photype A also caused more severe symptoms and could be recovered farther from the inoculation site than isolates of morphotype B, regardless of watering regime. These results indicate that water stress at levels observed typically in the field can result in increased disease development by isolates of S. sapinea morphotype A on red pine. The reduction of water stress of red pines in the field may reduce losses due to Sphaeropsis shoot blight.

Sphaeropsis sapinea and Water Stress in a Red Pine Plantation in Central Wisconsin.

ABSTRACT A study was conducted to determine the effects of water stress resulting from competing vegetation on disease development of Sphaeropsis sapinea in red pine plantations. A 9-year-old plantation was selected in 1992 and experiments were conducted for three consecutive years. Four treatments were assigned at random to individual trees: no treatment, herbicide to kill surrounding weeds, supplemental water, and both herbicide and supplemental water. Two isolates of each S. sapinea morphotype (A and B) were used to inoculate wounded lateral shoots. Disease development was measured as the maximum distance below the inoculation site at which necrotic needles were observed. Nonwatered trees with competing vegetation (nontreated condition) had significantly lower predawn needle water potentials (more water stress) and more severe disease development than trees that received the herbicide, water, or combined herbicide and water treatments. The most severe disease occurred in the driest year and the least in the wettest year. Competing vegetation indirectly affected disease development by inducing water stress, even in relatively moist years, on trees previously considered well established. Isolates of morphotype A were more aggressive than isolates of morphotype B. Conclusions from this research have implications for sustainable management of the region's conifer forests.

Sphaeropsis sapinea Morphotypes Differ in Aggressiveness, but Both Infect Nonwounded Red or Jack Pines.

Two morphotypes of Sphaeropsis sapinea, designated A and B, are recognized in the north central United States. Nonwounded seedlings of red (Pinus resinosa) and jack pine (P. banksiana) were inoculated with conidial suspensions of isolates of each morphotype obtained from hosts in Michigan, Minnesota, and Wisconsin. Wounded seedlings were inoculated with water agar plugs colonized by these isolates. Both morphotypes penetrated both hosts without wounding. On nonwounded seedlings the frequency of symptoms was 97% for A isolates and 18% for B isolates on red pine and 42% for A isolates and 6% for B isolates on jack pine. On average, isolates of the A morphotype also were more aggressive than B isolates on wounded seedlings of both pine species (A isolates causing needle necrosis 7.0 cm from the inoculation sites and B isolates 1.4 cm). Based on symptom severity (distance from the inoculation site at which necrotic needles were observed), A isolates could be distinguished from all B isolates on red pine, but not on jack pine. These observations indicate the potential importance of distinguishing between S. sapinea morphotypes encountered in nurseries, plantations, and natural stands.