ABSTRACT
Pumpkin (Cucurbita pepo L., cv. Magic Lantern) and watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai, cvs. Millionaire and Sangrea) plants with wilting leaves and collapse of entire vines were observed during the 2005 and 2006 growing seasons in several fields in southwestern New Mexico (Luna and Hidalgo counties) with an incidence ranging from 7 to 25% and less than 1% in pumpkin and watermelon fields, respectively. Sticky, hyaline strands were visible when vines were cut transversally, indicative of bacterial wilt caused by Erwinia tracheiphila (2). In the pumpkin fields, 12-spotted cucumber beetles, vector insects of E. tracheiphila, were found on plants at the first-true-leaf stage, which were treated with dimethoate. At the 4- to 5-leaf stage, 5 to 10% of the plants were wilted and were removed by hand. Less than 1% of the plants showed symptoms prior to bloom, when a high population of beetles was observed, and the fields were treated with thiamethoxam. To isolate the causal agent of the wilt symptoms, six, 1-cm vine segments and three to five beetles were surface sterilized in 1% NaOCl for 2 min, rinsed and macerated in sterile distilled water, and plated onto potato dextrose agar, nutrient agar, and King's medium B. After incubation at 25°C, bacterial colonies emerged on all media and were grayish white-to-cream, circular, smooth, and glittering. Isolated bacteria were gram negative, did not grow at 39°C, produced hydrogen sulfide gas from hydrolysis of cysteine, and did not hydrolyze litmus milk and starch. With Ready-To-Go PCR beads and 16S rDNA-based primers ET1/ET2 (1), a 700-bp product was obtained from each of two isolates, consistent with previously reported data for E. tracheiphila (1,3). For the pathogenicity tests, 10 seedlings of pumpkin cv. Magic Lantern and watermelon cv. Millionaire were inoculated with each isolate in the greenhouse at the second fully expanded leaf stage using two methods. In the first method, stems were injected with bacterial suspension (106 CFU/ml) using a hypodermic needle. In the second method, a dab of bacterial colonies was taken with a sterile toothpick to stab the cotyledonary axils. Control seedlings were stem injected with distilled water or stabbed with a sterile toothpick. The experiments were conducted four times. Inoculated plants were placed in a humid chamber at 23 to 25°C. Plant wilting was observed within 4 days when stab inoculated with toothpicks and within 7 to 10 days when stem injected with bacterial suspension. Bacterial colonies recovered from inoculated plants were identical to those recovered from field infected plants. To our knowledge, this is the first report of bacterial wilt on pumpkin and watermelon in New Mexico. References: (1) B. Bruton et al. Phytopathology (Abstr.) 89(suppl.):S10, 1999. (2) R. X. Latin. Page 36 in: Compendium of Curcurbit Diseases. T. A. Zitter et al., eds. The American Phytopathological Society, St. Paul, MN, 1996. (3) N. W. Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, 2001.
ABSTRACT
Head rot was found in cultivated sunflower (Helianthus annuus) in eastern New Mexico in Tucumcari in 2007 and Clovis in 2007 and 2009 and in south-central New Mexico near Las Cruces in 2009. The disease was also observed in wild sunflower near Clovis in 2008. Disease incidence was 10 to 40% in cultivated sunflower and ~30% in wild sunflower. Heads were brown to dark brown with discoloration extending down the sepals and peduncles into the stems. The basal parts of the heads were shredded and had grayish, fluffy mycelial mats visible in the lumen, and kernels were mostly seedless. Three to five diseased heads were collected from cultivated sunflower in 2007 and 2009 and wild sunflower in 2008. Plant tissues from heads and peduncles were surface sterilized for 3 min in 0.5% NaOCl, rinsed once in sterile distilled water, cut into 0.5-cm pieces, and plated on acidified potato dextrose agar (PDA). Within 3 to 7 days, mycelial colonies with abundant aerial growth and black sporangia emerged and were identified as Rhizopus oryzae on the basis of the presence of pale brown sporangiospores with bluish stripes (3) and mycelial growth at 36°C on PDA (1). PCR amplification of the internal transcribed spacer (ITS) region of rDNA from two isolates, one from cultivated and one from wild sunflower, using primer pair ITS4/ITS5 (1) was followed by sequencing and showed a 99% homology with the sequence of the ITS region of rDNA from R. oryzae (GenBank No. FJ654430). Each isolate was tested for pathogenicity on inflorescences (5 to 6 cm in diameter) of sunflower cvs. Hysun 511 and Triumph 820 HO grown for 4 to 5 weeks in a growth chamber at 26°C with a 14-h photoperiod. To obtain inoculum, a sterile toothpick was passed through a culture of R. oryzae until a ~3-mm mycelial mat was collected at the tip. The toothpick was dabbed into the center of an inflorescence or into the peduncle. A cotton boll was placed over the inoculation and sprayed with sterile distilled water. Control inflorescences were dabbed with toothpicks with no mycelium mat. Each inoculated and noninoculated inflorescence was covered with a plastic bag that was sealed around the peduncle. Plants were kept in the growth chamber for 3 weeks. In each of two experiments, 13 plants were used per cultivar and inoculation type, with 5 plants inoculated per isolate, and 3 control plants. Symptoms observed on inoculated sunflower were similar to those on field infected sunflower. There was no difference between the two cultivars. On inoculated inflorescences, dark discoloration developed at the inoculation site and expanded over the inflorescences, and grayish mycelium with black sporangia was observed within 2 weeks. On inoculated peduncles, dark discoloration was also observed extending down the peduncle and up into the inflorescences. R. oryzae was reisolated from all inoculated heads. To our knowledge, this is the first report of R. oryzae causing head rot on sunflower in New Mexico. It is unknown what factors lead to head rot outbreaks. This disease has been reported in other U.S. regions and has been demonstrated to reduce sunflower yield and quality (2). The potential negative impact from Rhizopus head rot should be considered when determining whether to expand cultivation of this crop. References: (1) G.-Y. Liou et al. Mycol. Res. 111:196, 2007. (2) C. E. Rogers et al. Plant Dis. Rep. 62:769, 1978. (3) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi: Morphologies of Cultured Fungi and Key to Species. CRC Press, Boca Raton, FL, 2002.
ABSTRACT
Wilted plants of Valencia market-type peanut (Arachis hypogaea L.) were found in two fields in August 2006 and three fields in September 2007 in Curry County, New Mexico. Plants had extensive, light brown discoloration and interstices of greenish tissue on blighted stems and branches across plant canopy levels. Disease incidence was less than 1% with infected plants in groups of two to five within each field. Five 4-cm stem segments were taken from each of five diseased plants in each field, submerged for 5 min in 0.5% NaOCl, rinsed in sterile distilled water, cut into 0.5-cm pieces, and plated on acidified potato dextrose agar (APDA). Mycelial colonies, recovered from plant tissues and incubated on APDA at 25°C under a 12-h photoperiod, were white and floccose with light green-yellow areas becoming visible within 7 to 10 days of incubation. Black stromata formed, spreading in a concentric pattern or scattered as large masses on APDA. Ostiolate and rostrate pycnidia with long beaks more than 500 µm were observed. Alpha conidia exuded from pycnidia in creamy-to-yellowish drops and were ellipsoid and biguttulate with an average length of 6.6 µm and width of 2.10 µm. Colonies were identified as Phomopsis longicolla Hobbs (1). PCR amplification of the internal transcribed spacer (ITS) region of rDNA of three isolates using primer pair ITS4/ITS5 (3) was followed by sequencing and BLAST analysis and showed a 95% homology with the sequence of the ITS region of rDNA of P. longicolla (1). Digestion of PCR-amplified DNA with AluI yielded two restriction fragments of sizes consistent with those reported for P. longicolla (2). Koch's postulates were established with three isolates tested for pathogenicity on Valencia peanut cv. Val-C at the four- to six-leaf stage using stem and root inoculations. Stems were injected with conidial suspension (106 conidia/ml) with a hypodermic needle or stabbed at the cotyledon axils with a sterile toothpick dabbed into an exuded conidial drop. Control plants were stem injected with distilled water or stabbed with a sterile toothpick. For root inoculation, plants were uprooted, washed free from soil, and inserted up to the crown into a 50-ml plastic test tube containing 40 ml of conidial suspension (25,000 conidia/ml) or sterile distilled water. For each method, eight plants were inoculated with each isolate, and four plants served as control. All inoculation methods were performed on the same day and repeated three times. Inoculated plants were covered with a clear plastic bag that was removed after 4 days. Plants were placed at 30°C under a 14-h photoperiod for 2 weeks. On stem-inoculated plants, light-to-dark brown discoloration formed at the sites of inoculation and expanded up and down the stems, which became brown, resulting in plant death within 10 to 14 days. On root-inoculated plants, browning of crown areas progressed up the stems, followed by plant death. P. longicolla was recovered from all inoculated plants. To our knowledge, this is the first report of P. longicolla on peanut in New Mexico and the United States. This report demonstrates the association of P. longicolla with peanut and its ability to cause stem blight. The occurrence and extent of this disease may be of a concern, because on other crops, Phomopsis diseases can cause significant reduction in seed germination, plant vigor, and yield. References: (1) T. W. Hobbs et al. Mycologia 77:535, 1985. (2) A. W. Zhang et al. Plant Dis. 81:1143, 1997. (3) A. W. Zhang et al. Phytopathology 88:1306, 1998.
ABSTRACT
Verticillium wilt, caused by Verticillium dahliae, is a common disease of chile pepper (Capsicum annuum) in New Mexico. In August of 2007, wilted plants with vascular discoloration in the stem typical of infection by V. dahliae occurred in several fields in Luna County in southern New Mexico. In one field, Verticillium wilt incidence was between 60 and 70%. Approximately 30% of the field was infested with Physalis wrightii (Wrights groundcherry) and Anoda cristata (spurred anoda), and 60% of the field was infested with Ipomoea purpurea (tall morningglory). Except for vascular discoloration found in a few plants of Wrights groundcherry and spurred anoda, there were no other symptoms observed in the weeds present. Previously, Wrights groundcherry and spurred anoda were demonstrated as hosts to V. dahliae (2); however, to our knowledge, tall morningglory was not. A 5-cm segment was cut from the lower part of the stems and upper part of the tap roots of six tall morningglory plants and two chile pepper plants. The segments were washed, surface disinfested for 2 min in 0.5% sodium hypochlorite, and cut into pieces that were plated onto water agar. Mycelial colonies emerging from the pieces were transferred to either potato dextrose agar, prune extract agar, or Czapek-Dox agar medium. Putative V. dahliae isolates from tall morningglory and chile pepper plants were identified based on characteristic morphological features when cultured on prune extract medium (2,3). In addition, PCR of fungal DNA and sequencing the amplicons using primer pair ITS4/ITS6 showed a 99% homology with the sequence of the rDNA ITS of V. dahliae (1). Pathogenicity tests were conducted with two isolates of V. dahliae from tall morningglory and one from chile pepper. In the first of two methods, four pots were infested with conidia of each isolate (2 × 107 conidia per 500 cm3 of soilless mix) and planted (five seeds per pot, thinned to three seedlings) with chile pepper cv. AZ-20, which is susceptible to V. dahliae. Three noninfested pots served as the control. Pots were placed in a growth chamber at 26/20°C day/night temperature. In the second method, plants (cv. AZ-20) at the 6- to 8-leaf stage were inoculated in a greenhouse with V. dahliae by dispensing 5 ml of a conidial suspension (4 × 106 conidia/ml) into the root plug prior to transplanting. Four root plugs were inoculated per isolate and there were three noninoculated root plugs. Both experiments were repeated once. Isolates of V. dahliae recovered from tall morningglory and chile pepper were pathogenic on chile pepper. Leaf chlorosis, leaf drop, wilting, and vascular discoloration were observed within 8 weeks after sowing into infested soil or within 6 weeks after inoculation into the root plugs of transplants. No symptoms were observed on noninoculated plants. V. dahliae was reisolated from the stems of all symptomatic plants. To our knowledge, this is the first report to document the recovery of V. dahliae from tall morningglory and its pathogenicity on chile pepper. References: (1) P. V. Pramateftaki et al. J. Fungal Genet. Biol. 29:19, 2000. (2). S. Sanogo and M. Clary. Plant Dis. 87:450, 2003. (3) P. W. Talboys. Plant Pathol. 9:57, 1979.
