Differences in Virulence and Sporulation of Phytophthora kernoviae Isolates Originating From Two Distinct Geographical Regions.

Phytophthora kernoviae has only been isolated from the United Kingdom and New Zealand. To understand what differences may exist between isolates from these two distinct geographical regions, virulence studies on three host plants and sporulation on host leaves were conducted on select isolates. Three host plant species (Rhododendron ponticum, Magnolia stellata, and Annona cherimola) were inoculated individually with sporangia of six different isolates from each geographical region. Results showed an overall higher virulence on all three hosts from isolates originating from the United Kingdom. After inoculation, P. kernoviae sporangia and oospore formation on different host leaves were observed and compared with P. cactorum and P. syringae. Results were host dependent, with P. kernoviae producing generally similar or higher amounts of both propagules compared with the other U.S. indigenous species. These results have implications for regulatory agencies and scientists who are interested in preventing its entrance into the United States and learning more about its potential spread.

Infectivity and Inoculum Production of Phytophthora ramorum on Roots of Eastern United States Oak Species.

Little is known about colonization of roots of trees by Phytophthora ramorum. We examined zoospore concentration and exposure time needed to infect six Quercus (oak) species and the inoculum produced from their roots. Sprouted acorns, exposed to zoospores (3,000/ml) for different times and transplanted to potting soil, were susceptible to infection within 1 h of exposure but root weights were not impacted after 4 weeks (P = 0.952). Roots of Quercus prinus seedlings, inoculated with sporangia, had 0.6 to 3.2% colonization of the total root mass after 5 months. Neither root lesions nor obvious root sloughing were observed. Inoculum threshold levels were tested by exposing radicles to varying zoospore concentrations for 24 h. Results showed that radicle infection occurred even at 1 zoospore/ml. To test inoculum production, roots were inoculated with sporangia and transplanted into pots. Periodically, samples of runoff were collected and plated on selective medium. Afterward, root segments were plated to calculate percent colonization. After 16 and 35 days, root colonization and inoculum production from oak was lower than that of Viburnum tinus, a positive control. This study shows that P. ramorum is able to infect sprouted oak acorns and produce secondary inoculum, which may be important epidemiologically.

Field Damage to Yellow Starthistle Infected by Synchytrium solstitiale, and Greenhouse Maintenance and Host Range of the Fungus.

Yellow starthistle (YST, Centaurea solstitialis) is a major weed pest of the western United States. Synchytrium solstitiale, a pathogen of YST, caused significant damage to symptomatic (versus asymptomatic) plants in a field study in France. Before it was evaluated as a candidate for biological control of YST in the United States, protocols for pathogen maintenance under greenhouse conditions were developed. Maintenance, increase, and host range determination protocols involved incubation at 10/15°C (night/day) with an 8-h photoperiod either of potted or exhumed (i.e., roots of 4-week-old plants grown in flasks of water) plants inoculated with galled leaf tissue, or potted plants in which inoculum was wrapped within healthy leaves by a plastic wrap. The leaf-wrap protocol, used during the host range determination, always resulted in disease of YST. Several safflower (Carthamus tinctorius) cultivars and other plants related to YST became diseased following this protocol, thus raising concern about host specificity. Development of disease on nontarget species precludes proposal of S. solstitiale for biological control of YST at this time.

Infective Potential of Sporangia and Zoospores of Phytophthora ramorum.

Phytophthora species produce sporangia that either germinate directly or release zoospores, depending upon environmental conditions. Previous Phytophthora spp. inoculation trials have used both sporangia and zoospores as the inoculum type. However, it is unknown what impact propagule type has on disease. Rhododendron leaf disks were inoculated with P. ramorum zoospores (75, 500, or 2,400 per disk), sporangia (75 per disk), or sporangia plus trifluoperazine hydrochloride (TFP) (75 per disk), a chemical that inhibits zoospore formation. Combining results from two different isolates, the highest concentration of zoospores (2,400 per disk) induced a significantly higher percentage of necrotic leaf disk area (96.6%) than sporangia (87.6%) and 500 zoospores per disk (88.7%). The sporangia plus TFP treatment had the lowest necrosis at 47.5%. Rooted rhododendron cuttings had a higher percentage of necrotic leaves per plant when inoculated with zoospores (3,000 or 50,000 per ml) or cysts (50,000 per ml) than with sporangia (3,000 per ml) with or without TFP. The percentage of necrotic leaf area was significantly higher when cysts or zoospores were inoculated at 50,000 per ml than sporangia without TFP and zoospores at 3,000 per ml. All treatments were significantly higher in the percentage of necrotic leaf area than the leaves treated with sporangia plus TFP. This demonstrates that the full inoculum potential may not be achieved when sporangia are used as the inoculum propagule.

An IPM Program for Managing Fungal Leaf Blight Diseases of Carrot in New York.

Fungal leaf blight diseases caused by Cercospora carotae and Alternaria dauci occur annually on processing carrot in New York, with growers applying up to eight fungicide sprays to manage these diseases. An integrated pest management (IPM) program involving the use of a 25% disease incidence threshold to prompt the first fungicide application and timing subsequent sprays by monitoring for increases in disease severity and weather forecasts in conjunction with a 10- to 14-day spray interval was evaluated in grower fields in 1997 and 1998. The IPM plots, compared with the grower plots, required two to six fewer fungicide applications but showed no yield reduction. From 1999 to 2004, the IPM program was validated and the effect of crop rotation and carrot cultivar susceptibility also were assessed. Carrot plants growing in fields with 2-year or longer crop rotation intervals reached the 25% disease incidence threshold later in the season and required fewer fungicide applications. The less-susceptible carrot cultivars also reached the 25% disease incidence threshold later, required fewer fungicide applications, and were less severely diseased than more susceptible cultivars. Validation of the IPM program in New York showed that both fungal leaf blights can be managed effectively using a 25% incidence threshold to prompt the first fungicide spray and making the subsequent fungicide applications based on increases in disease severity, weather forecasts, and a 10- to 14-day spray interval.

Factors Affecting Infection of Yellow Starthistle (Centaurea solstitialis) by Synchytrium solstitiale, Causal Agent of False Rust Disease.

Yellow starthistle (Centaurea solstitialis) is an annual invasive weed in the United States with Mediterranean origins. The expense of chemical control and the vast area of invasion make this weed an appropriate target for classical biological control. Observations of a field site in southern France revealed small orange galls on the leaves of yellow starthistle seedlings caused by the fungus Synchytrium solstitiale. Inoculation of yellow starthistle seedlings with a suspension of zoospores released from infected tissue resulted in infection. Ten days after inoculation, typical orange galls appeared on the exposed tissue. Preliminary host range testing showed up to 100% infection of C. solstitialis seedlings from both France and the United States and infection of Carthamus tinctorius, Centaurea americana, C. diffusa, C. rothrockii, C. squarrosa, and Helianthus annuus seedlings. No symptoms were observed on seedlings of Centaurea calcitrapa, C. maculosa, C. sulfurea, Cirsium californica, C. occidentale, Cynera cardunculus, and Taraxacum officinale. Zoospores were released in a pH range between 4.45 and 8.25 and optimally at temperatures between 5 and 15°C. Infection of yellow starthistle seedlings occurred after a minimum 1-h exposure to a zoospore suspension at 20°C.

Cryopreservation of Synchytrium solstitiale In Planta.

The fungus Synchytrium solstitiale is a candidate for use as a biocontrol agent against Centaurea solstitialis. This obligate parasite can be propagated only in planta, which necessitates development of a method for preserving cultures for longer periods of time for routine biological studies and shipment to other laboratories. Normally, sporangia embedded within the plant tissue release zoospores when submersed in water at temperatures above freezing. To examine what chemicals might inhibit zoospore release, infected tissue was exposed to different suspensions of fungicides. Cycloheximide and benomyl completely inhibited zoospore release or immediately induced encystment from tissue stored in these two chemicals, respectively. A few zoospores were released in suspensions of iprodione and propionic acid but were not motile. However, when tissue stored in iprodione or propionic acid was transferred to fresh distilled water, abundant active zoospores were released. Freezing the infected tissue at different temperatures in different cryoprotectants also affected the release of motile zoospores. Infected C. solstitialis tissue was immersed in water, water plus iprodione, methanol, ethylene glycol, dimethyl sulfoxide, glycerol, skim milk, trehalose, or sucrose and subjected to different temperatures for various periods of time. Some treatments protected the viability of the fungus for a shorter period of time whereas other treatments completely inhibited release. The best results were obtained when infected tissue was stored at -2°C in 0.5 M sucrose where active zoospores were released after 12 weeks of storage. Overall, results obtained from this study demonstrate a technique for long-term storage of S. solstitiale.

Relationship Between Levels of Cyanide in Sudangrass Hybrids Incorporated into Soil and Suppression of Meloidogyne hapla.

Sudangrass cv. Trudan 8 has been demonstrated to suppress infection of vegetables by Meloidogyne hapla (Mh). Hydrogen cyanide, released from the degradation of the cyanogenic glucoside (dhurrin) during decomposition of Trudan 8, was the primary factor involved in suppression of Mh on vegetables. The cyanide ion level in leaf tissue of 14 hybrids of sudangrass varied between 0.04 (cv. SX-8) to 1.84 parts per million (cv. 840F). The suppressive activity of the sudangrass hybrids against Mh was assessed in greenhouse tests by incorporating various amounts of leaf tissue into organic soil. After 1 week, eggs of Mh were added to the soil (8 eggs/cm[sup3] soil), which was then planted with lettuce as a bioassay plant. After 8 weeks, the lettuce roots were washed and rated for root-gall severity (RGS). Incorporation of sudangrass tissue resulted in a reduction of RGS up to 54%. There was a correlation between the amount of free cyanide incorporated into the soil and the reduction in RGS. Other green manures of cyanogenic plants tested were white clover, which resulted in a 45% reduction in RGS, and flax, which resulted in a 53% reduction in Mh penetration of lettuce roots. These results suggest that cyanogenic plants have potential as nematicidal green manures.

Soil organic matter and management of plant-parasitic nematodes.

Organic matter and its replenishment has become a major component of soil health management programs. Many of the soil's physical, chemical, and biological properties are a function of organic matter content and quality. Adding organic matter to soil influences diverse and important biological activities. The diversity and number of free-living and plant-parasitic nematodes are altered by rotational crops, cover crops, green manures, and other sources of organic matter. Soil management programs should include the use of the proper organic materials to improve soil chemical, physical, and biological parameters and to suppress plant-parasitic nematodes and soilborne pathogens. It is critical to monitor the effects of organic matter additions on activities of major and minor plant-parasitic nematodes in the production system. This paper presents a general review of information in the literature on the effects of crop rotation, cover crops, and green manures on nematodes and their damage to economic crops.

Mechanism of Suppression of Meloidogyne hapla and Its Damage by a Green Manure of Sudan Grass.

Meloidogyne hapla is a continuing problem on vegetables in the state of New York. Sudan grass has been demonstrated to suppress infection and damage to susceptible vegetables caused by this nematode when incorporated as a green manure. Some Sudan grass cultivars contain the cyanogenic glucoside dhurrin that is degraded through an intermediate step to p-hydroxybenzal-dehyde (p-HBA) and HCN. Incubating M. hapla eggs in Sudan grass extract resulted in a 55% reduction in the number of juveniles (J2) penetrating lettuce roots. Juveniles exposed to the extracts were not affected. Sudan grass extract affected egg maturation by delaying development, but did not affect hatching. Exposing eggs to a 0.1 ppm CN- solution reduced the number of nematodes that penetrated the roots by 48%, while exposing J2 to the same concentration reduced nematode infection by only 4%. However, exposure of eggs to p-HBA did not reduce J2 penetration into roots. After purifying Sudan grass extract through size-exclusion resin, only the fractions collected that contained cyanide suppressed infection of lettuce roots by M. hapla J2 when eggs were exposed to these fractions. These results suggest that CN- is the primary factor involved in the suppression of M. hapla by a green manure of Sudan grass.

Composted Municipal Waste Reduces Infection of Citrus Seedlings by Phytophthora nicotianae.

Most citrus nurseries and orchards in Florida are infested with Phytophthora nicotianae, the causal agent of Phytophthora root rot. Although fungicides control the disease and increase seedling growth and tree yields, they are not always economically or environmentally sustainable. Amendment of citrus soils with composted municipal waste (CMW) may provide an alternative to fungicides for disease management. Citrus seedling growth decreased with increasing proportions of one CMW source, indicating the potential for phytotoxicity from soluble salts and acetic acid when the proportion of CMW in soil exceeded 20% (vol/vol). When a citrus soil was amended (20% vol/vol) with certain sources of CMW, the incidence of infection of 5-week-old susceptible citrus seedlings by P. nicotianae was reduced from 95% to as low as 5%. Addition of fresh CMW to two different citrus soils reduced colony growth of P. nicotianae after the fungus was incubated in the amended soil for 6 days and then recovered on PARPH selective medium. If CMW was stored for more than 3 months before amendment, the soil mixtures did not suppress colony development. In general, extracts of fresh CMW reduced mycelial growth in vitro; whereas suppressiveness of CMW from the same batch was lost after storage. Acetic acid was detected at higher concentration in fresh CMW that suppressed colony growth than in CMW that lacked activity. A species of Acremonium was isolated from another source of CMW that was suppressive to P. nicotianae. This fungus parasitized hyphae of P. nicotianae in vitro. Thus, CMWs have the potential to at least temporarily suppress P. nicotianae through chemical and microbial agents depending on source and age of the CMW.

Histological Comparison of Fibrous Root Infection of Disease-Tolerant and Susceptible Citrus Hosts by Phytophthora nicotianae and P. palmivora.

ABSTRACT Phytophthora nicotianae and P. palmivora infect and cause rot of fibrous roots of susceptible and tolerant citrus rootstocks in Florida orchards. The infection and colonization by the two Phytophthora spp. of a susceptible citrus host, sour orange (Citrus aurantium), and a tolerant host, trifoliate orange (Poncirus trifoliata), were compared using light and electron microscopy. Penetration by both Phytophthora spp. occurred within 1 h after inoculation, regardless of the host species. No differences were observed in mode of penetration of the hypodermis or the hosts' response to infection. After 24 h, P. palmivora had a significantly higher colonization of cortical cells in susceptible sour orange than in tolerant trifoliate orange. Intracellular hyphae of both Phytophthora spp. were observed in the cortex of sour orange, and cortical cells adjacent to intercellular hyphae of P. palmivora were disrupted. In contrast, the cortical cells of sour orange and trifoliate orange adjacent to P. nicotianae hyphae and the cortical cells of trifoliate orange adjacent to P. palmivora were still intact. After 48 h, the cortical cells of both hosts adjacent to either Phytophthora spp. were disrupted. After 48 and 72 h, P. palmivora hyphae colonized the cortex of sour orange more extensively than the cortex of trifoliate orange; P. palmivora also colonized both hosts more extensively than P. nicotianae. A higher rate of electrolyte leakage among host-pathogen combinations reflected the combined effects of greater cell disruption by P. palmivora than by P. nicotianae, and the higher concentration of electrolytes in healthy roots of trifoliate orange than of sour orange. Although cellular responses unique to the tolerant host were not observed, reduced hyphal colonization by both pathogens in the cortex of trifoliate orange compared with sour orange is evidence for a putative resistance factor(s) in the trifoliate orange roots that inhibits the growth of Phytophthora spp.