Fusarium Wilt Disease Control Using Biological Agents Trichoderma and Mycorrhizaeon Pepper

Fusarium oxysporum is the main fungus disease that can wither plants, especially pepper. The fungus spread through diseased soil or already withered plants and then infect plants from its roots. Doing control by using antagonistic fungi such as Trichoderma sp. and Arbuscularmycorrhizae have been widely performed. Trichoderma sp. is a fungus rich with an antifungal activity that produce metabolites, both volatile and non-volatile. These metabolites produced by Trichoderma can diffuse through the dialysis membrane which capable to slow several pathogens growth. Mycorrhizae creates mutual symbiosis between certain types of fungi with roots, also known as biological agents, capable to control F. oxysporum on pepper and to help antibiotics formation. The study was conducted at Laboratorium Hama dan Penyakit BPTP East Java, starting from January to May 2016. This study used completely randomized design (CRD) with treatment consisted of 16 combined doses of Mycorrhizae and Trichoderma, each repeated 4 times that produce 64 test units. Mycorrhizae dose used is 0.0; 1.0; 2.0 and 4.0 grams per polybag, while the Trichoderma dose used is 0.0; 15.0; 30.0; and 45.0 grams per polybag. Data were statistically analyzed by variance analysis and followed by a BNT test of 0.05. The results showed Mycorrhizae 4 g /polybag and Trichoderma 45 g /polybag application could increase the incubation period of F. Oxysporum fungus, shorten xylem discoloration and then reduce wilted plants percentage. Mycorrhizae application can boost Trichoderma fungus in order to reduce wilt disease found in pepper plants

Pathogenicity of Ganoderma Species on Landscape Trees in the Southeastern United States

The genus Ganoderma contains species that are associated with dead and declining host trees. Many species have been described as pathogens in literature, because anecdotally, the presence of fruiting bodies on living trees has been widely associated with a general decline in tree health. Few studies have investigated the pathogenicity of Ganoderma species on landscape trees in the southeastern U.S. Pathogenicity tests were used to determine the pathogenicity of G. curtisii, G. meredithiae, G. sessile, and G. zonatum on young, healthy landscape trees (Pinus elliottii var. elliottii, P. taeda, Quercus shumardii, Q. virginiana, and Butia odorata) common to the southeastern U.S. Inoculations were made by drilling into the sapwood of the lower bole and inserting wooden dowels that were infested with each Ganoderma species. In two field experiments, 11 to 12 months post inoculation, trees had no visual, external symptoms of disease. There were differences in the extent of internal xylem discoloration near the site of inoculation in comparison with the mock-inoculated control in experiment 1, but there were no differences relative to the control in experiment 2. In both experiments, G. sessile was the only species that was successfully reisolated from the pine and oak hosts. Although disease symptoms were not obvious, the reisolation of G. sessile outside the inoculation point was a significant finding, and suggests that this species was capable of infecting healthy sapwood. G. sessile constitutively produces chlamydospores within its vegetative mycelium, which may contribute to its persistence in the discolored sapwood. These data suggest that the Ganoderma species tested, following trunk wounding, are not pathogens of young, actively growing landscape trees that only possess sapwood. The establishment of these fungi using alternative infection courts (e.g., roots) and their interactions in older living trees (e.g., trees with heartwood) needs investigation to better understand their effects on tree health.

First Report of Laurel Wilt, Caused by Raffaelea lauricola, on Sassafras (Sassafras albidum) in Alabama

Laurel wilt, caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, is responsible for extensive mortality of native redbays (Persea borbonia and P. palustris) in the coastal plains of the southeastern United States (1). The wilt also affects the more widespread sassafras, Sassafras albidum, particularly in areas where diseased redbays are common and populations of X. glabratus are high. Because sassafras stems were thought to lack chemicals that are attractive to the beetle, and sassafras tends to be widely scattered in forests, it was believed that the advance of the laurel wilt epidemic front might slow once it reached the edge of the natural range of redbay, which is restricted to the coastal plains of the Gulf and Atlantic Coasts (2). In July and August of 2011, wilt-like symptoms (i.e., wilted and dead leaves, and streaks of black discoloration in the xylem) were observed on 1 to 10 sassafras trees (15 to 23 cm diameter; 6 to 9 m height) at each of three locations, which were approximately 6 km from one another in Marengo Co., Alabama. Samples of the discolored wood from five trees were plated on malt agar amended with cycloheximide and streptomycin (CSMA), and a fungus morphologically identical to R. lauricola was isolated from each tree (1). For confirmation, a portion of the large subunit (28S) of the rDNA region of three of the isolates was sequenced (3); in each case, the sequence matched exactly that of other isolates of R. lauricola (EU123077) from the United States. Symptomatic trees were found at all three sites when revisited in April 2012, and approximately 20 sassafras trees in various stages of wilt were observed at one location, where only one diseased tree had been noted in 2011. Bolts were cut from the main stem of a symptomatic tree, and eggs, larvae, and adults of X. glabratus were commonly found in tunnels, and R. lauricola was isolated from the discolored xylem. Three container-grown sassafras saplings (mean height 193 cm, mean diameter 2.1 cm at groundline) were inoculated as previously described (1) with conidia (~600,000) from an isolate of R. lauricola. Three additional sassafras saplings were inoculated with sterile, deionized water, and all plants were placed in a growth chamber at 25°C with a 15-h photoperiod. Inoculated plants began to exhibit wilt symptoms within 14 days, and at 30 days all inoculated plants were dead and xylem discoloration was observed. Control plants appeared healthy and did not exhibit xylem discoloration. Pieces of sapwood from 15 cm above the inoculation points were plated on CSMA, and R. lauricola was recovered from all wilted plants but not from control plants. This is the first record of laurel wilt in Alabama and is significant because the disease appears to be spreading on sassafras in an area where redbays have not been recorded (see http://www.floraofalabama.org ). The nearest previously documented case of laurel wilt is on redbay and sassafras in Jackson Co., Mississippi (4), approximately 160 km to the south. The exact source of the introduction of X. glabratus and R. lauricola into Marengo Co. is not known. The vector may have been transported into the area with storms, moved with infested firewood, or shipped with infested timber by companies that supply mills in the area. References: (1) S. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. Hanula et al. Econ. Ent. 101:1276, 2008. (3) T. Harrington et al. Mycotaxon 111:337, 2010. (4) J. Riggins et al. Plant Dis. 95:1479, 2011.

First Report of Bark Cracking of Koelreuteria bipinnata var integrifoliola Caused by Lasiodiplodia theobromae in China

Koelreuteria bipinnata var integrifoliola is becoming a popular urban green tree in Ningbo City, Zhejiang Province, China, because of its adaptation ability to local conditions, fast growth, and beautiful appearance. A survey conducted from 2007 to 2010 revealed serious bark cracking on greenbelt trees approximately 15 to 16 years old that had been transplanted 5 to 6 years ago. Bark cracks increased in size over time, extending into the phloem and leading to extensive areas of bark loss with discoloration of the underlying xylem. Symptomatic trees had fewer new shoots in spring; many wilted and died in summer. Root rot was not observed in the withered trees but large light brown lesions were observed on cross sections of the main stem, each with a dark brown outer margin. In a September 2009 survey, 95% of symptomatic trees had stem lesions more than 50 cm long. Pieces of xylem (2 × 2 × 1 mm thick) were obtained from the margin of lesions surface sterilized using 0.1% mercuric chloride for 30 s, washed in sterile distilled water, and placed on 2% potato dextrose agar (PDA) at 28°C for 2 days. The fungus was then isolated and 12 colonies were obtrained. Three isolates KL-1-2, KL-3-2, and KL-4-3 were incubated on 2% PDA at 28°C for 30 days to produce spores. On PDA, the colonies were circular or near circular with irregular gray edges turning black green or black. The fungus also produced abundant aerial hyphae that were villous, septate, and irregular branched. Conidia were elliptical (or rounded) and hyaline when immature, becoming dark brown and septate longitudinally when mature and ranged from 23.2 to 27.0 × 10.8 to 16.2 μm (average 25.3 × 13.6 μm), similar to Lasiodiplodia theobromae (Patouillard) Griffon =Botryodiplodia theobromae Pa.t, Botryosphaeria rhodina (Berkeley & Curtis) von Arx (2). DNA extraction directly from the mycelium of KL-1-2, KL-3-2, and KL-4-3 was performed after 10 days' growth on PDA (1). The identities of the three isolates were confirmed by ITS1-5.8S-ITS2 rDNA sequence (GenBank Accession Nos. JN681172, JQ894322, and JQ894323, respectively) analysis that showed 99%, 100%, and 100% sequence similarity to L. theobromae xsd08006 (Accession No. FJ478102), L. theobromae PD20 (Accession No. GU251120), and L. theobromae xsd08008 (Accession No. EU918707), respectively. Pathogenicity tests were performed on 20 five-year-old K. bipinnata var integrifoliola plants by placing mycelia plugs of isolate KL-1-2 (10 × 10 mm) on the main trunk after wounding with a metal needle. Control plants received PDA plugs without mycelium. After inoculation, humidity was maintained using wet absorbent cotton and PE wrap film. Stem bark and phloem cracking was observed after 60 days on 85% of inoculated plants; 30% of those trees also had xylem discoloration. Symptoms were similar to those with natural infection. Control plants remained symptomless. The same fungus was reisolated from the brown xylem of inoculated plants. To our knowledge, this is the first report of bark cracking of K. bipinnata var integrifoliola caused by L. theobromae in China. References: (1) M.-J. Côté et al. Plant Dis. 88:1219, 2004. (2) G. Fu et al. Australas. Plant Dis. Notes 2:75, 2007.

New Insights into Esca of Grapevine: The Development of Foliar Symptoms and Their Association with Xylem Discoloration

A new study on the development of foliar symptoms of esca was carried out from 2004 to 2006 in five mature vineyards in Aquitaine, France. Symptoms were monitored for severity and changes over time. Initial foliar symptoms were characterized by the presence of drying zones or discolorations (reddening or yellowing), which are symptoms that have also been attributed to Black Dead Arm (BDA). Then, the less-severely affected leaves persisted throughout the summer and developed into typical “tiger-stripe” symptoms of esca. The most severely symptomatic leaves fell soon after symptoms appeared. Severely diseased vines showed typical apoplectic or acute forms of esca that did not differ from the severe BDA forms. The appearance of leafsymptomatic vines increased uniformly over time, reaching a maximum incidence by the end of July. A second survey in 41 European and Lebanese vineyards showed that longitudinal discolorations were visible under the bark of 95% of the vines showing foliar esca symptoms. These wood symptoms, also previously attributed to BDA, appeared as xylem orange-brown stripes. Thus, foliar symptoms of esca showed transitory phases which overlapped with some BDA descriptions. Most of these symptoms, in the west-palearctic regions that were investigated, were commonly associated with the presence of one or several xylem discolorations.

First Report of Chalara fraxinea on Common Ash in Italy

In many European countries, the anamorphic Chalara fraxinea Kowalski (teleomorph Hymenoscyphus albidus [Roberge ex Desm.] Phillips; 1–3) is responsible for a severe and rapidly spreading dieback of common ash (Fraxinus excelsior L.) since it was first reported in Poland. Recently, this disease was added to the EPPO Alert List and the NAPPO Phytosanitary Alert System. Symptomatic trees were observed in a 1.8-ha ash-maple forest in northeastern Italy (Fusine, UD; 46°30′N, 13°37′E; 782 m above sea level) along the Italo-Slovenian border in July 2009. Symptoms were found on approximately 10% of mature common ash and 70% of seedlings. Main symptoms were shoot, twig, and branch dieback, wilting, and bark cankers (1). Fungal fruiting bodies were not found on or near the canker surface. Furthermore, longitudinal and radial sections through the cankers revealed gray-to-brown xylem discoloration. One symptomatic 3-year-old plant was randomly selected and from the necrotic margin of one canker previously surface-sterilized with 3% sodium hypochlorite and rinsed, four 2-mm-wide chips were placed on malt extract agar (MEA) and incubated at 21 ± 1°C in the dark. Among a variety of microorganisms, after 19 days, slow-growing colonies (mean radius of 12 mm) appeared that were effuse, cottony, and often fulvous brown but sometimes dull white with occasional gray-to-dark gray patches. The purified isolate was then transferred to the same medium at 4 ± 1°C in the dark, and after 11 days, hyaline-to-dark gray phialides were observed producing numerous conidia in slimy droplets and sometimes in chains. Phialophores measured 8.6 to 21.0 (15.1) μm long (n = 20), 4.2 to 13.4 (8.8) × 3.6 to 5.5 (4.7) μm at the base, and 5.2 to 8.7 (6.5) × 2.5 to 3.1 (2.8) μm at the collarette; conidia measured 2.8 to 4.2 (3.4) × 1.9 to 2.5 (2.2) μm (n = 40); and first formed conidia measured 5.5 to 6.5 (5.9) × 1.8 to 2.5 (2.1) μm (n = 20). These morphological characteristics matched Kowalski's (1) description of C. fraxinea. In August of 2009, the fungal isolate was used to test pathogenicity with current year shoots of 25 6-year-old (150 to 210 cm high) asymptomatic common ash trees under quarantine conditions (Slovenian Forestry Institute's experimental plots). For every plant, the bark of the main shoot (10 to 13 mm in diameter) was wounded with a 6-mm-diameter cork borer. Twenty saplings were inoculated with one 6-mm-diameter mycelial plug obtained from the margin of a 26-day-old culture (MEA), while five saplings were inoculated with sterile MEA plugs. All wounds were sealed with Parafilm and aluminum foil. After 28 days, all plants inoculated with the C. fraxinea showed bark lesions (2 to 39 mm long, mean 7 mm) and wood discoloration (6 to 85 mm long, mean 22 mm) from which the pathogen was reisolated. These symptoms were absent from controls and the pathogen was never reisolated. To our knowledge, this is the first report of C. fraxinea in Italy. Investigations on its presence in all Fraxinus species naturally growing in the investigated area and in the nearest regions are in progress. The obtained isolate is preserved in both Padova and Ljubljana herbaria as CFIT01. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:304, 2009.

Resistance of Eucalyptus Clones to Ceratocystis fimbriata

Ceratocystis fimbriata, the inciting agent of wilt, canker, and dieback in eucalyptus plantations, was first reported in Brazil in 1998. There is no information regarding the resistance of Eucalyptus spp. to this pathogen. We determined the reaction of 18 Brazilian, commercial clones of the hybrid Eucalyptus grandis × E. urophylla to inoculation by two isolates of the pathogen in two experiments. Container-grown, 8-month-old rooted cuttings of each clone were wound-inoculated with a conidial suspension (2.5 × 106/ml). Plants similarly injected with sterile distilled water served as controls. The plants were evaluated after 30 days for length of xylem discoloration. There was a significant isolate by clone interaction, but most clones reacted similarly to the two isolates. Six clones, including one observed to be highly susceptible under field conditions, were highly susceptible (>20 cm discoloration) to one or both isolates of the pathogen. Four clones showed no more discoloration with either isolate than with the control inoculations, and the other eight clones were intermediate in susceptibility. Thus, highly resistant clones are available to manage this disease.

Characterization and Distribution of Two Races of Phialophora gregata in the North-Central United States

Genetic variation and variation in aggressiveness in Phialophora gregata f. sp. sojae, the cause of brown stem rot of soybean, was characterized in a sample of 209 isolates from the north-central region. The isolates were collected from soybean plants without regard to symptoms from randomly selected soybean fields. Seven genotypes (A1, A2, A4, A5, A6, M1, and M2) were distinguished based on DNA fingerprinting with microsatellite probes (CAT)5 and (CAC)5, with only minor genetic variation within the A or M genotypes. Only the A1, A2, and M1 genotypes were represented by more than one isolate. The A genotypes dominated in the eastern Iowa, Illinois, and Ohio samples, whereas the M genotypes were dominant in samples from western Iowa, Minnesota, and Missouri. In growth chamber experiments, isolates segregated into two pathogenicity groups based on their aggressiveness toward soybean cvs. Kenwood and BSR101, which are relatively susceptible and resistant, respectively, to brown stem rot. In both root dip inoculation and inoculation by injecting spores into the stem near the ground line (stab inoculations), isolates of the A genotypes caused greater foliar symptoms and more vascular discoloration than isolates of the M genotypes on both cultivars of soybean. All isolates caused foliar symptoms in both cultivars and in three additional cultivars of soybean with resistance to brown stem rot. Greater differences between the A and M genotypes were seen in foliar symptoms than in the linear extent of xylem discoloration, and greater differences were seen in Kenwood than in BSR101. Inoculation of these genotypes into five cultivars of soybean with different resistance genes to brown stem rot showed a genotype × cultivar interaction. A similar distinction was found in an earlier study of the adzuki bean pathogen, P. gregata f. sp. adzukicola, and consistent with the nomenclature of that pathogen, the soybean pathogens are named the aggressive race (race A) and the mild race (race M) of P. gregata f. sp. sojae.

First Report of Ceratocystis Wilt of Acacia mearnsii in Uganda

Ceratocystis albofundus, the cause of Ceratocystis wilt of Acacia mearnsii, is known only from South Africa. The only known hosts of this fungus are A. mearnsii, Acacia decurrens, and two species of Protea (1). This pathogen causes stem cankers, xylem discoloration, wilt, and the death of susceptible A. mearnsii trees in South Africa, leading to considerable losses to the forestry industry (1). During a recent survey of forest plantation diseases in Uganda, A. mearnsii trees with “streaked” discoloration of the xylem, typical of Ceratocystis infection, were found in southwestern Uganda. These trees had been damaged mechanically by the harvesting of side branches and/or stems for firewood and construction. Xylem discoloration was spreading through the trees from these wounds. Trees showed typical stem cankers and gummosis, which is associated with C. albofundus infection, as well as foliage wilting. Isolations from infected trees yielded a fungus that was similar morphologically to C. albofundus, with typical hat-shaped ascospores and light-colored perithecial bases (2). Sequencing of the internal transcribed spacer region of the ribosomal RNA operon of Ugandan isolates (CMW5329, CMW5964, GenBank accession no. AF388947) confirmed their identification, grouping them with C. albofundus and separating them from all other Ceratocystis species. This is the first report of C. albofundus from a country other than South Africa. C. albofundus is an important pathogen, and strategies to reduce losses need to be established in Uganda because the aggressiveness of C. albofundus to A. mearnsii has been shown in inoculation experiments (1). References: (1) Morris et al. Plant Pathol. 42:814, 1993. (2) Wingfield et al. Syst. Appl. Microbiol. 19:191, 1996.

Sporulation of Fusarium oxysporum f. sp. lycopersici on Stem Surfaces of Tomato Plants and Aerial Dissemination of Inoculum

Plants exhibiting symptoms of wilt and xylem discoloration typical of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici were observed in greenhouses of cherry tomatoes at various sites in Israel. However, the lower stems of some of these plants were covered with a pink layer of macroconidia of F. oxysporum. This sign resembles the sporulating layer on stems of tomato plants infected with F. oxysporum f. sp. radicis-lycopersici, which causes the crown and root rot disease. Monoconidial isolates of F. oxysporum from diseased plants were assigned to vegetative compatibility group 0030 of F. oxysporum f. sp. lycopersici and identified as belonging to race 1 of F. oxysporum f. sp. lycopersici. The possibility of coinfection with F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was excluded by testing several macroconidia from each plant. Airborne propagules of F. oxysporum f. sp. lycopersici were trapped on selective medium in greenhouses in which plants with a sporulating layer had been growing. Sporulation on stems was reproduced by inoculating tomato plants with races 1 and 2 of F. oxysporum f. sp. lycopersici. This phenomenon has not been reported previously with F. oxysporum f. sp. lycopersici and might be connected to specific environmental conditions, e.g., high humidity. The sporulation of F. oxysporum f. sp. lycopersici on plant stems and the resultant aerial dissemination of macroconidia may have serious epidemiological consequences. Sanitation of the greenhouse structure, as part of a holistic disease management approach, is necessary to ensure effective disease control.