DIALLEL ANALYSIS OF RESISTANCE TO BACTERIAL STALK ROT (Pectobacterium chrysanthemi pv. zeae Burk., McFad. and Dim.) IN CORN (Zea mays L.)

One of the major disease problems affecting maize farming in the Philippines is bacterial stalk rot (BSR) caused by Pectobacterium chrysanthemi pv. zeae Burk., McFad. and Dim., which is formerly known as Erwinia chrysanthemi. The annual loss due to BSR is estimated at PhP. 20 million equivalent to Rp3.5 billion. At present, there is no effective control method against BSR and, therefore, varietal improvement through breeding resistant germplasms is needed. The present study aimed to determine the combining ability and the extent of additive and non-additive genetic effects in corn inbred lines with a range of reaction to BSR. Four resistant lines (S3YB 137-1-1-B, TUPI (S3) 5-1-B, TUPI (S3) 15-2-B, and 97-835) and two susceptible lines (CML 295 and 97-733) were used as genetic materials. Generation of test entries and evaluation of disease resistance were conducted at the experimental farm station of University of the Philippines Los Banos and Institute of Plant Breeding Los Banos, respectively, during 2002 to 2003 wet seasons. Griffing’s diallel mating system Model 1, Method 1 was followed in generating the test entries to make a total of 36 entries (six selfed parental lines and 15 each of F1 crosses and their reciprocal<br />crosses). The entries were then evaluated for disease resistance in a yield trial following a randomized complete block design (RCBD) with two replications. Results of diallel analysis<br />showed two lines, S3YB 137-1-1-B and TUPI (S3) 5-1-B, exhibited the best general combining ability (GCA) for resistance to BSR, while the crosses S3YB 137-1-1-B x TUPI (S3) 5-1-B and TUPI (S3) 5-1-B x 97-835 performed the best specific combining ability (SCA) for the resistance. GCA effect was greater than that of SCA. This indicated that additive gene effects were found to be more important than non-additive gene effects in the expression of resistance to BSR in the six corn lines used. Therefore, breeding programs towards recurrent selection that emphasize GCA would be more appropriate for<br />BSR resistance improvement involving those six lines.

DIALLEL ANALYSIS OF RESISTANCE TO BACTERIAL STALK ROT (Pectobacterium chrysanthemi pv. zeae Burk., McFad. and Dim.) IN CORN (Zea mays L.)

One of the major disease problems affecting maize farming in the Philippines is bacterial stalk rot (BSR) caused by Pectobacterium chrysanthemi pv. zeae Burk., McFad. and Dim., which is formerly known as Erwinia chrysanthemi. The annual loss due to BSR is estimated at PhP. 20 million equivalent to Rp3.5 billion. At present, there is no effective control method against BSR and, therefore, varietal improvement through breeding resistant germplasms is needed. The present study aimed to determine the combining ability and the extent of additive and non-additive genetic effects in corn inbred lines with a range of reaction to BSR. Four resistant lines (S3YB 137-1-1-B, TUPI (S3) 5-1-B, TUPI (S3) 15-2-B, and 97-835) and two susceptible lines (CML 295 and 97-733) were used as genetic materials. Generation of test entries and evaluation of disease resistance were conducted at the experimental farm station of University of the Philippines Los Banos and Institute of Plant Breeding Los Banos, respectively, during 2002 to 2003 wet seasons. Griffing’s diallel mating system Model 1, Method 1 was followed in generating the test entries to make a total of 36 entries (six selfed parental lines and 15 each of F1 crosses and their reciprocal<br />crosses). The entries were then evaluated for disease resistance in a yield trial following a randomized complete block design (RCBD) with two replications. Results of diallel analysis<br />showed two lines, S3YB 137-1-1-B and TUPI (S3) 5-1-B, exhibited the best general combining ability (GCA) for resistance to BSR, while the crosses S3YB 137-1-1-B x TUPI (S3) 5-1-B and TUPI (S3) 5-1-B x 97-835 performed the best specific combining ability (SCA) for the resistance. GCA effect was greater than that of SCA. This indicated that additive gene effects were found to be more important than non-additive gene effects in the expression of resistance to BSR in the six corn lines used. Therefore, breeding programs towards recurrent selection that emphasize GCA would be more appropriate for<br />BSR resistance improvement involving those six lines.

Bacterial Soft Rot of Oncidium Orchids Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in Florida

Oncidium orchids have been subjected to extensive cultivation in the pot-plant and cut flower industries because of their attractive and numerous flowers. In August 2008, approximately 50 Oncidium ‘Gower Ramsey’ orchids were discovered at a commercial orchid nursery in South Florida with brown, macerated leaves typical of soft rot disease reported in other orchids. Ten plants were selected, and sections were removed from the edge of symptomatic tissue and bacteria were isolated according to the method described by Schaad et al. (3). All isolates were gram negative, anaerobic, degraded pectate, grew at 37°C, produced blue-to-brown pigment on nutrient agar-glycerol-manganese chloride (NGM) medium (1), were sensitive to erythromycin, oxidase negative, and positive for phosphatase and indole production. Further analyses were performed on four of the isolates. MIDI analysis (Sherlock version TSBA 4.10; Microbial Identification, Newark, DE) identified the isolates as Erwinia chrysanthemi (SIM 0.880 to 0.929). Polymerase chain reactions were performed with the 16S primers 27f and 1495r (4) and 1,423 bp of the 16S rDNA gene showed 98 to 99% sequence identity to Pectobacterium chrysanthemi (GenBank Accession No. FM946179). Sequences were deposited in GenBank (Nos. HQ287572–HQ287575). Pathogenicity tests were performed by injecting 10 Oncidium ‘Gower Ramsey’ orchids with 100 μl of a bacterial suspension at 1 × 108 CFU/ml. Ten plants were inoculated with 100 μl of sterile water as controls. Plants were placed in a greenhouse at 26.0°C to 30.0°C and 50 to 83% relative humidity. Soft rot symptoms were observed on all inoculated plants within 24 h while control plants appeared normal. A Dickeya sp. was reisolated and identified according to the method described above. Oncidium orchids are known to be highly susceptible to P. carotovora (= E. carotovora) and soft rot caused by P. carotovora is known to occur frequently on Oncidium orchids (2). Although, an Erwinia sp. has been reported to cause soft rot symptoms on Oncidium aureum, to our knowledge, this is the first report of a Dickeya sp. (= P. chrysanthemi) causing soft rot symptoms on Oncidium orchids grown in large-scale commercial production in the United States. References: (1) Y. A. Lee and C. P. Yu. J. Microbiol. Methods 64:200, 2006. (2) C. H. Liau et al. Transgenic Res. 12:329, 2003. (3) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of a Soft Rot of Banana in Mainland China Caused by a Dickeya sp. (Pectobacterium chrysanthemi)

Banana is one of the most important fruit crops grown in China (2). A severe outbreak of a soft rot of banana occurred in Guangzhou, China from 2009 to 2010. The disease was characterized by an odorous soft rot of the center of the rhizome. The rot progressed up the pseudostem, destroying the growing point and causing internal decay and often accompanied by vascular discoloration. Yellowing and wilting of the leaves were also characteristic symptoms. A survey of three areas of production of Musa sapientum (cv. ABB) covering 10 ha in Guangzhou revealed that 82% of the fields were affected at an incidence ranging from 20 to 40%. Forty-five bacterial isolates were obtained from lesions on plants sampled from these fields by surface-sterilizing symptomatic tissue in 0.3% NaOCl for 10 min, rinsing the tissue sections three times in sterile water, and plating the sections on nutrient agar. Three representative isolates selected randomly were all gram negative, caused a soft rot of potato disks, utilized malonate, tested positive for phosphatase production, and tested negative for acid production from palatinose, glucopyranoside, and trehalose. A Biolog similarity index of 0.803 indicated that the three isolates had a high similarity to the Biolog reference strain of Pectobacterium chrysanthemi (Version 4.2, Biolog Inc., Hayward, CA). The 16S rDNA sequence (GenBank Accession No. 1321085) of each of the three isolates was determined (1). When compared with sequences in GenBank, the highest degree of sequence similarity was with P. chrysanthemi AF373199. On the basis of a phylogenetic tree of the sequences, the three bacterial isolates are related to Pectobacterium (100% bootstrap values). On the basis of two diagnostic methods, the three isolates were identified as P. chrysanthemi. However, according to Samson et al. (3), they are a Dickeya sp. Additional genetic comparisons with type strains will be needed for the strains to be assigned to a known species of Dickeya. Pathogenicity of each of the three strains on M. sapientum (cv. ABB) was confirmed by injecting 60 40-day-old seedlings each with 5 ml of a suspension of the isolate (108 CFU/ml) into the rhizome. Sixty plants of the same cultivar injected with sterile water served as the control treatment. After 48 h, yellowing and wilting of the leaves, similar to symptoms observed on field plants, were observed on all inoculated seedlings for each of the three bacterial strains. There were no symptoms on the control plants. Koch's postulates were fulfilled by reisolating bacteria from lesions on the leaves of inoculated seedlings. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of a Dickeya sp. causing soft rot of banana in mainland China. References: (1) W. S. Kaneshiro et al. Plant Dis. 92:1444, 2008. (2) Y. P. Ke et al. China Trop. Agric. 1:14, 2008. (3) R. Samson et al. Evol. Microbiol. 55:1415, 2005.

Bacterial Stem Rot of Oncidium Orchid Caused by a Dickeya sp. (ex Pectobacterium chrysanthemi) in Mainland China

In December 2007, stem rot symptoms on orchids (Oncidium Gower Ramsey) were observed at a flower nursery in the Zhejiang Province of China. Initial symptoms were water-soaked lesions starting at the base of the stem. As these lesions expanded and elongated, the stem and leaf tissues became soft and watery. When examined with a microscope, cut edges of symptomatic stem and leaf tissues consistently exhibited bacterial streaming. The bacteria were isolated by streaking on nutrient agar (3). All isolates were gram-negative, facultative, anaerobic rods with peritrichous flagella. Infiltration of tobacco leaves (Nicotiana tabacum cv. Samsun) with the bacterial suspension of 108 CFU/ml resulted in typical hypersensitivity reactions within 24 h. Five representative isolates were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) by the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TSBA50). The five isolates were identified as Erwinia chrysanthemi (Pectobacterium chrysanthemi) with a Biolog and FAME similarity index of 0.81 to 0.88 and 0.62 to 0.75, respectively. The transfer of P. chrysamthemi to a novel genus, Dickeya gen. nov., was recently proposed (2). The almost complete 16S rDNA sequence from Oncidium isolate SCH-01 (1,604 bp; EMBL Accession No. FM946179) was determined according to the method of Li et al. (1). A subsequent GenBank search showed that this isolate is 98% identical to that of type strain CFBP 1269T of Dickeya dadantii (EMBL Accession No. AF520707) and CFBP 1200T of Dickeya dianthicola (EMBL Accession No. AF520708). Nevertheless, species identification within genus Dickeya is still difficult since only a limited number of strains of each species have been characterized fully. Koch's postulates were completed with the inoculation of Oncidium seedlings with cell suspensions (108 CFU/ml) by a pinprick at the base of the stem. All five representative isolates induced stem rot similar to that observed in natural infections. No symptoms were noted on the control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of Oncidium plants. To our knowledge, this is the first report of stem rot on Oncidium orchid in Mainland China caused by the bacterium formerly referred to as P. chrysanthemi, now proposed as Dickeya sp. References: (1) B. Li et al. J. Phytopathol. 154:711, 2006. (2) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

Bacterial Stem Rot of Poinsettia Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in China

In December 2006, a rot symptom of unknown etiology was observed on stems of plants (Euphorbia pulcherrima cv. Fu-xing) at a flower nursery in the Zhejiang Province of China where we had previously reported leaf spot of poinsettia caused by Xanthomonas campestris (2). Chlorotic spots anywhere along the stem and purplish black petioles were the first noticeable symptoms. The spots rapidly coalesced, forming large irregular chlorotic areas. Petioles turned black and shriveled and affected leaves wilted. Infected tissues were soft and water soaked. Ten bacterial strains were isolated from the diseased samples and five were selected for identification. They were similar to those of the standard reference strains of Pectobacterium chrysanthemi (Dickeya sp.), LMG 2804 from Belgium and ZUPB20056 from China, in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with aerobic bacterial library (TABA50), and transmission electron microscopy (TEM,KYKY-1000B, Japan). All strains tested were gram-negative facultative anaerobic rods measuring 1.5 to 3.6 × 0.6 to 1.1 μm, with peritrichous flagella. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They were negative for trehalose and positive for phosphatase production and reducing substances from sucrose. A hypersensitive reaction was observed on tobacco cv. Benshi, 24 h after inoculation. All five isolates, LMG 2804, and ZUPB20056 were identified as P. chrysanthemi (Dickeya sp.) with a Biolog similarity index of 0.58 to 0.83, 0.68, and 0.72 and a FAME similarity index of 0.52 to 0.80, 0.59, and 0.70, respectively. Identification as P. chrysanthemi (Dickeya sp.) was confirmed by PCR with specific primers used by Nassar et al (3). Koch's postulates were completed with the inoculation of 12 4-month-old intact poinsettia plants of cv. Fu-xing with cell suspensions containing 108 CFU/ml by a pinprick at the base of the stem. All five strains induced stem infection similar to those observed in natural infections. No symptoms were noted on the two control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of poinsettia plants. P. chrysanthemi (Dickeya sp.) was first reported in United States as the cause of bacterial stem rot of poinsettia in 1972 (1). To our knowledge, this is the first report of poinsettia stem rot caused by P. chrysanthemi (Dickeya sp.) in China. The disease cycle and the control strategies of the bacterial stem rot of poinsettia in the regions are being further studied. References: (1) H. A. J. Hoitink et al. Plant Dis. Rep. 56:480, 1972. (2) B. Li et al. Plant Pathol. 55:293, 2006. (3) A. A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996.