Soft Rot Lesions Research Articles

First report of Pseudomonas viridiflava causing fruit rot on sweet cherry trees in Chile.

Chile is an important producers of sweet cherry (Prunus avium L.), with a total of 356,385 t exported in the 2021 to 2022 season. The production area includes most of the country's regions. Bacterial samples were isolated in 2017 and 2018 from 18 commercial sweet cherry orchards with canker disease. From one of this samples collected in the spring of 2018, was isolated the strain A2M176 from buds of trees that presented canker and gomosis in Malloa locality (34°23' 46'' S 71°01' 39'' W). The strain produced fluorescent pigment on King's B agar medium. Is oxidase and arginine dihydrolase negative, potato soft rot positive and showed a slight degree of tobacco hypersensitivity. It was able to growth up to 0.8 mM (200 ppm) of CuSO4·5H2O. The strain A2M176 was deposited in the Colección Chilena de Recursos Genéticos Microbianos (CChRGM) under the no. RGM 3342. The DNA of this strain was extracted from a pure culture using silica spin columns (Epoch Life Science Inc., Sugar Land, USA). The complete DNA was sequenced using HiSeq with 150 bp paired-end at GENEWIZ (New York, USA). Raw data was checked using FASTQC and trimmed with BBDuk. The genome was assembled using Unicycler v0.4.9 with defaulf settings and annotated with Prokaryotic Genome Annotation Pipeline (PGAP) v4.3. The reads and genomes were uploaded to GenBank under the BioProyect no. PRJNA750090, BioSample no. SAMN26870984 and assembly no. GCA_022936465.1. The sequenced genome was compared through Average Nucleotide Identity algorithm (ANI) using FastANI v1.33 to compare with closest complete genome available on NCBI. The strain A2M176 was identified as P. viridiflava with ANI value of 98.06% with the strain p22.E7 (GCF_900585495). Maximum likelihood phylogenetic estimation clustered strain A2M176 with other P. viridiflava strains with 95% bootstrap. The pathogenicity of the strain was tested inoculating immature cherry fruits with a needle with a bacterial suspension (1x108 CFU/ml). The inoculated fruits were placed at room temperature in a humid chamber for 10 d. Soft rot lesions were observed, which appeared at 6 days post-inoculation (DPI). The control fruits treated with sterile water did not show symptoms. Further analyses in the genome of strain A2M176 led to identify genes related to pathogenicity, such as the effector gene avrE and the regulator gen HrpL, suggesting the pathogenic capacity of the strain. Also, there were identify genes of two known Pseudomonas copper resistance mechanisms, the cus and cop operon. These genes were found part of the copABCDns cluster similar to what was observed in Pseudomonas from Mango. Presence of P. viridiflava strains causing fruit rot in P. avium is not surprising, since P. viridiflava has a wide host range and causes a variety of symptoms in different plant parts, including stems, leaves, and blossoms. P. viridiflava represents one of the multiple phylogroups found within the P. syringae species complex. To our knowledge, this is the first report of a strain of P. viridiflava copper resistant causing infection on sweet cherries in Chile.

Soft rot disease caused by Dickeya fangzhongdai in epiphytic orchids in Vietnam

Soft rot diseases are becoming problematic in the cultivation of epiphytic orchids in Vietnam, but the causal agents are unknown. We investigated soft rot in Dendrobium anosmum, Phalaenopsis amabilis and Paphiopedilum concolor. Leaves of diseased plants with soft rot symptoms were sampled and cultures of Dickeya were obtained. The pathogen was identified as Dickeya fangzhongdai based on a phylogenetic analysis of 16s rRNA, dnaX, gapA and rplB. The pathogenicity of nine D. fangzhongdai isolates was confirmed in D. anosmum, Ph. amabilis and Pa. concolor in a nursery trial. The efficacy of a range of chemical agents was evaluated on the growth of D. fangzhongdai in vitro, and on D. fangzhongdai soft rot disease management in 1-year-old D. anosmum, Ph. amabilis and Pa. concolor plants. Cuprous oxide and Cu-bismerthiazol strongly inhibited the in vitro growth of D. fangzhongdai, and they reduced the soft rot lesion area of infected plants in vivo. These findings provide an interim option for producers to improve management of D. fangzhongdai soft rot disease in production nurseries in Vietnam.

First Report of Dickeya fangzhongdai causing soft rot in Orchids in Canada.

Dickeya fangzhongdai was originally described as the causal agent of bleeding canker of pear tree in China. Recently, D. fangzhongdai was isolated and identified as the causal agent of soft rot in an orchid plant purchased in a local supermarket in Prince Edward Island, Canada. A water-soaked dark green spot on the leaf surface was observed and later became larger soft rot symptom. The origin of the orchid plants was traced back to a producer in Ontario, Canada who propagated them from with cuttings originally imported from the Netherlands and Taiwan. Bacterial isolations were made from a soft rot lesion on an orchid leaf by surface sterilization of small pieces of marginal tissue of the diseased leaf in 70% alcohol. The small pieces of leaf tissue were then washed three time using sterile water, and immersed in drops of sterile water. Bacterial streaming was observed under the microscope and non-fluorescing bacterial colonies were isolated on King's B and casamino acid-peptone-glucose agar plates and purified as isolates 908, 909, 910 and 911. The DNA samples were extracted from the four isolates, as well as the diseased leaf tissue, and tested by using a qPCR assay with the specific primer/probe set (DfF/DfR/DfP) for D. fangzhongdai (Tian et al. 2020). The assay yielded PCR amplicons of 135 bp with a melting temperature of 86.5±0.6 °C as did two control reactions using genomic DNA from D. fangzhongdai strains JS5T and QZH3 originally isolated in China, providing presumptive identification of the orchid isolates as D. fangzhongdai. To fulfill Koch's postulates, freshly purchased healthy orchid plants (n=4) were inoculated by leaf injection with the bacterial isolates obtained in this study and strains JS5 T and QZH3 at ~107 CFU/ml. Three leaves of the same side of the plants were inoculated with the same strains as triplicates. Sterile water was used as the negative control. Inoculated plants were incubated in a growth chamber with a 16 h photoperiod at 23 °C. Water soaked lesions developed in 3-5 days after inoculation followed by soft rotting in leaves inoculated with the new bacterial strains from orchid plants while strain QZH3 caused soft rot in 10 days after inoculation (Fig. S1). The non-fluorescing bacteria on King's B plates with colony morphology similar to those inoculated were re-isolated from the inoculated leaves and confirmed to be D. fangzhongdai by qPCR. Phylogenetic analysis of the assembled 16S rRNA sequence of isolate 908 (GenBank accession number: MT984340), together with GenBank data of all Dickeya spp. and some Pectobacterium spp, using neighbor-joining (NJ) method inferred with MEGA X software (Kumar et al. 2018) showed that isolate 908 clustered with strains JS5T and QZH3 at a phylogenetic distance of 0.0007. This clearly indicated that isolate 908 and JS5T and QZH3 belong to the same genus. Species-level identification of isolate 908 was achieved by genome sequencing and analysis based on average nucleotide identity (ANI). Genomic DNA of isolate 908 was sequenced with Illumina MiSeq to provide approximately 180X genome coverage. After quality checking using FastQC (Andrews 2010), de-novo assembly was performed with VelvetOptimiser v2.2.6 (Zerbino and Birney 2008). The draft genome size of strain 908 was 4,938,027 bp consisting of 76 contigs with 56.8% G+C content and 63,801 bp as N50. The draft genome was checked for misassembled fragments using QUAST v5.0.2 (Gurevich et al. 2013) and found to be of good quality. The draft genome sequence is deposited in GenBank under the accession number of JADCNJ000000000. The draft genome sequence of strain 908 was compared to that of D. fangzhongdai JS5T type strain genome using FastANI v1.2 (Jain et al. 2018) resulting in an ANI value of 98.9%, which is above the 95% cut-off for the same species. Previously, it was reported that D. fangzhongdai caused soft rot in orchid in Europe (Alič et al. 2018) and in onions in New York (Ma et al. 2020). The difference in virulence among D. fangzhongdai strains warrants further investigation and their pathogenicity on potato is being investigated to evaluate any threat to the potato industry. To our knowledge, this is the first report of D. fangzhongdai causing soft rot disease on orchids in Canada and North America.

Detection of Lesions in Lettuce Caused by Pectobacterium carotovorum Subsp. carotovorum by Supervised Classification Using Multispectral Images

This study aimed to detect soft rot caused by Pectobacterium carotovorum subsp. carotovorum in lettuce using images obtained by multispectral sensors mounted on an unmanned aerial vehicle (UAV). A secondary objective was to identify the best sensor and determine the optimal stage after inoculation to detect infected plants. In the field, soft rot lesions and the agronomic traits of lettuce plants inoculated or not with the bacteria were assessed on different days after inoculation (DAI). Classifications were made using the Support Vector Machine (SVM) and Naive Bayes (NB) algorithms to analyze data groups consisting of spectral bands, vegetation indices and a combination of bands and indices obtained from a conventional visible camera and Mapir Survey3W multispectral camera, as well as agronomic parameters. The results confirmed the possibility of pre-symptomatic detection of P. carotovorum subsp. carotovorum in lettuce at the canopy level. With respect to identifying healthy and infected lettuce plants by supervised classification, the best results were obtained at 4 and 8 DAI, especially when using the subsets derived from the Mapir Survey3W camera (RGN sensor), for both classifiers. The subsets obtained with the conventional visible sensor (RGB sensor) produced the best results at 20 and 24 DAI.

Pseudomonas allii sp. nov., a pathogen causing soft rot of onion in Japan.

Six phytopathogenic bacterial strains, MAFF 301512, MAFF 301513, MAFF 301514T, MAFF 301515, MAFF 301516 and MAFF 301517, were isolated from soft rot lesions of onion (Allium cepa L.) in Japan. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella and rod-shaped. Analysis of their 16S rRNA gene sequences showed that they belong to the genus Pseudomonas, with the highest similarities to Pseudomonas poae DSM 14936T (99.86 %), Pseudomonas simiae OLiT (99.85 %), Pseudomonas trivialis DSM 14937T (99.79 %) and Pseudomonas extremorientalis KMM 3447T (99.79 %). Their genomic DNA G+C content was 60.9 mol% and the major fatty acids (>5 % of the total fatty acids) present were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c) and C17 : 0 cyclo. Phylogenetic and phylogenomic analyses based on the rpoD gene and whole genome sequences, respectively, demonstrated that the strains belong to the Pseudomonas fluorescens subgroup, but form a monophyletic and robust clade, with Pseudomonas azotoformans as their neighbour. Between the strains and P. azotoformans, the average nucleotide identity scores were 95.63-95.70 %, whereas the digital DNA-DNA hybridization scores of the strains against their closest relatives, including P. azotoformans, were 65.4 % or less, which are lower than the 70 % cut-off for prokaryotic species delineation. The strains were differentiated from their closest relatives by phenotypic characteristics, pathogenicity in onion and cellular fatty acid composition. The phenotypic, chemotaxonomic and genotypic data showed that the strains represent a novel Pseudomonas species, proposed to be named Pseudomonas allii sp. nov., with MAFF 301514T (=ICMP 23680T) being the type strain.

First Report of Plantain Soft Rot Caused by Klebsiella variicola in Haiti

Plantain and banana, both Musa spp. hybrids, constitute a major component of domestic food production in Haiti. In response to grower reports (Faculty of Agronomy and Veterinary Medicine, Port-au-Prince, personal communication) of up to 60% incidence of “toppling disease”, we conducted a preliminary study in July 2018 at four plantain farms in Arcahaie, Haiti. Symptoms observed on up to 30% of plants (n = ∼100) included a brown necrotic soft rot lesion on pseudostems extending up to 1 m in length and several centimeters deep. Toppling occurred at fruit onset, with plants folding over at the point of the soft rot. Six plants representing the four farms were sampled: 6-cm² sections were excised from the lesion edge, surface sterilized with 0.6% sodium hypochlorite, rinsed, and macerated with sterile tap water, and then the suspension was streaked onto nutrient agar (NA) and Miller-Schroth medium agar (MSM) plates, yielding six bacterial isolates. Colonies were round, glossy, convex, translucent gray-white on NA, produced a transient red-orange color change on MSM, and were aerobic, gram-negative, oxidase-negative and hypersensitive response negative on ‘Bonnie Best’ tomato and ‘Hicks’ tobacco (Schaad et al. 2001); all the above tests were repeated twice. All six isolates were identical in initial 16S rDNA sequencing, so pathogenicity of two randomly chosen isolates (G18-1365 and G18-1376) was tested on disease‐free ‘Dwarf Cavendish’ banana plantlets by drilling a 2.5-cm hole into the pseudostem, inserting sterile cotton-tipped swabs drawn through 48-h cultures on NA (negative control was dipped in sterile water) into the drilled incision, and sealing with Parafilm. Each plant was wounded once, with five plants per isolate or control. Rotting symptoms consistent with the field observations were observed 4 weeks after inoculation for all isolate inoculations; control plants remained symptomless. Reisolation and identification to fulfill Koch’s postulates were completed with results identical to the first isolations and tests. Genomic DNA was boil-extracted as previously described (Dashti et al. 2009) from the two isolates grown 24 h on NA. The amplification and sequencing of 16S rDNA, rpoB, phoE, and infB gene targets were carried out using primers and protocols previously described (Rosenblueth et al. 2004). One sequence for each isolate’s gene target (eight total) was deposited in NCBI GenBank (accessions MK217521 to MK217522; MK235993 to MK235998). In BLAST searches, sequence fragments of 16S rDNA, rpoB, phoE, and infB from isolates G18-1365 and G18-1376 resulted in 99% identity with Klebsiella variicola strains. Multilocus sequence analysis (MEGA X, Kumar et al. 2018) of those sequences yielded a concatenated phylogenetic tree that placed both isolates within the K. variicola clade. Isolates G18-1365 and G18-1376 have been deposited at the BCCM, Ghent University (accessions LMG 31122 and LMG 31123, respectively). K. variicola has been reported to cause similar bacterial rots in banana and carrot (Chandrashekar et al. 2018; Fan et al. 2016). Before management can be prescribed, further studies are necessary to determine disease incidence and if this species is the sole cause of toppling or is part of a larger disease complex, and to understand the mode of transmission within and among plantain farms. Additionally, similarity to human pathogenic strains should be studied to determine if there is a human health risk posed by K. variicola isolates causing soft rot in plantain.

BACTERIAL SOFT ROT OF EGGPLANT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN CHINA

In December 2016, soft rot lesions on the stem and fruit of eggplants cv. Green Beauty were noted in greenhouses in Anshan, Liaoning Province, China. Disease incidence was estimated at 5-10% and resulted in a substantial yield reduction. Symptoms appeared as soft, dark green stem lesions that progressively turned brown with vascular bundle necrosis appearing. Fruits of infected plants initially had small water-soaked lesions that rapidly enlarged with a watery ooze. Infected tissues from stems and fruits of eggplant were macerated in sterile water, streaked onto nutrient agar (NA) and incubated at 28oC for 2 days. The resultant bacterial colonies were round, translucent, white, convex with smooth edges. All isolated bacteria were gram-negative rods, facultative anaerobes, non-fluorescent on King’s B media, produced a positive pectolytic response on crystal violet pectate agar and gave a positive hypersensitive response in tobacco plants. All were negative for sucrose reduction, sensitive to erythromycin (50 µg/ml), indole, oxidase and could grow at 37°C. PCR amplification of the pel gene using Y1/Y2 primers (Darrasse et al., 1994) produced a 430 bp fragment. Amplification of the 16S- 23SrRNA region by G1 and L1 primers (Toth et al., 2001) produced two main bands of 550 and 580 bp. 16S rRNA fragment of 1440 bp was amplified from bacterial isolates and the partial sequence deposited in GenBank (accession No. KY786125) showed 99% identity to the sequence of Pectobacterium carotovorum subsp. carotovorum strain Y57 (KP187518.1). No amplification products were produced for any isolate using P. atrosepticum and P. carotovorum subsp. brasiliensis specific primers (Eca1f/Eca2r and Br1f/L1r, respectively), whereas the P. carotovorum subsp. carotovorum primers EXPCCF/EXPCCR produced the expected 550 bp fragment (Kang et al., 2003). For pathogenicity testing, mature fruits and stems of Green Beauty were needle-stab inoculated with a 10 µl bacteria suspension (1×108 CFU/ml). Sterile water was used as a negative control. Inoculated plants were incubated at 28°C for 48 h, after which watersoaked and soft-rot symptoms were observed on all eggplant fruits and stems, but not on control plants. Bacteria with the same characteristics as those inoculated were reisolated from diseased tissues. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum on eggplant in China.

Paromomycin Derived from Streptomyces sp. AG-P 1441 Induces Resistance against Two Major Pathogens of Chili Pepper.

This is the first report that paromomycin, an antibiotic derived from Streptomyces sp. AG-P 1441 (AG-P 1441), controlled Phytophthora blight and soft rot diseases caused by Phytophthora capsici and Pectobacterium carotovorum, respectively, in chili pepper (Capsicum annum L.). Chili pepper plants treated with paromomycin by foliar spray or soil drenching 7 days prior to inoculation with P. capsici zoospores showed significant (p < 0.05) reduction in disease severity (%) when compared with untreated control plants. The disease severity of Phytophthora blight was recorded as 8% and 50% for foliar spray and soil drench, respectively, at 1.0 ppm of paromomycin, compared with untreated control, where disease severity was 83% and 100% by foliar spray and soil drench, respectively. A greater reduction of soft rot lesion areas per leaf disk was observed in treated plants using paromomycin (1.0 μg/ml) by infiltration or soil drench in comparison with untreated control plants. Paromomycin treatment did not negatively affect the growth of chili pepper. Furthermore, the treatment slightly promoted growth; this growth was supported by increased chlorophyll content in paromomycin-treated chili pepper plants. Additionally, paromomycin likely induced resistance as confirmed by the expression of pathogenesis-related (PR) genes: PR-1, β-1,3-glucanase, chitinase, PR-4, peroxidase, and PR-10, which enhanced plant defense against P. capsici in chili pepper. This finding indicates that AG-P 1441 plays a role in pathogen resistance upon the activation of defense genes, by secretion of the plant resistance elicitor, paromomycin.

Characterization of the Causal Organism of Soft Rot of Tomatoes and Other Vegetables and Evaluation of Its Most Aggressive Isolates

To isolate the causal organism of soft rot of vegetables, diseased samples of potato, tomato, carrot, chilies, and bell pepper, were analyzed in the lab, using nutrient agar (NA) and/or the enrichment host (Bell pepper) technique. Successful isolations were purified by sub-culturing, identified as Erwinia carotovora subsp. carotovora through biochemical tests and their pathogenicity was confirmed through inoculation on green tomato fruits. The isolates were tested for their aggressiveness to find out the most aggressive one in term of producing maximum soft rot on tomatoes. CRD (completely randomized design), with four replication was used and data were analyzed using LSD (least significant test) test. Among the five isolates evaluated for aggressiveness on tomato fruits, chili isolate was found to be the most aggressive followed by tomato and potato isolates producing 22.3 mm, 7.9 mm, and 7.8 mm diameter soft rot lesions, respectively.

First Report of Pineapple Black Rot Caused by Ceratocystis paradoxa on Ananas comosus in French Guiana.

Ceratocystis paradoxa (Dade) C. Moreau is a polyphagous wound parasite causing black rot post-harvest disease in pineapple. This fungus is responsible for high losses of fruit destined for the fresh market and is a common problem in many countries (2). C. paradoxa is officially listed as a quarantine pathogen for French Guiana. In November 2013, the Plant Protection Service of French Guiana observed damage in crops of Ananas comosus var. perolera located in Corossony (4°19'10.8″ N, 52°10'17.1″ W) and Wayabo (5°01'02.3″ N, 52°36'18.7″ W) (eastern and middle part of the country). The three plants collected at each location showed a soft base rot of the stem and of young leaves, and developed a foul smell. Plant tissues were collected from the edge of the lesions, chopped in small pieces, and plated on malt extract agar supplemented with 100 ppm chloramphenicol. The plates were incubated at 25°C with a 12-h photoperiod. After 5 days, a fungal colony, first white and downy, then becoming black and velvety after 10 days, was transferred on potato dextrose agar (PDA) and incubated in the same conditions. After 7 days, the colonies produced phialides releasing cylindrical or doliform conidia that were unicellular, colorless to pale brown, in long chains (3.09 to 20.17 × 3.1 to 5.57 μm, n = 20) and oval, pyriform, brown chlamydospores (8.02 to 21.32 × 4.20 to 9.76 μm, n = 20), occurring in long chains or singly with a vertical slit, usually not very visible. Furthermore, the colonies emitted a fruity odor. On the basis of these morphological characteristics, the fungus was identified as the anamorph of C. paradoxa (Thielaviopsis paradoxa (De Seynes) Höhn.) (1). The species designation was confirmed by sequencing the ITS region of the rDNA followed by comparison with reference sequences available in GenBank. Fungal material was collected from PDA culture by scraping the mycelium with a sterile needle and transferring into 2-ml microtubes. Fungal total DNA was then extracted and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair. Nucleotide sequence was determined and deposited in GenBank (KJ667047). BLAST analysis showed 100% identity with C. paradoxa. The pathogenicity of the fungus was confirmed by inoculating two pineapples with mycelium from the C. paradoxa isolate grown on PDA. The peel of fruits and the base of the crowns were wounded with a sterile scalpel blade, each at five locations. Mycelial plugs (avg. 4 mm diameter) were placed on the wounds. Inoculation sites were wrapped with Parafilm to prevent dehydration and to hold the mycelial plugs in position. Negative controls received five sterile PDA plugs. The samples were incubated at 25°C in a moist chamber with a 12-h photoperiod. Eight days after inoculation, negative controls remained symptomless, whereas characteristic soft, watery, and black rot lesions developed on the base of all the crowns that were inoculated with C. paradoxa. The pathogen was successfully re-isolated from symptomatic tissues, fulfilling Koch's postulate. To our knowledge, this is the first report of C. paradoxa on A. comosus in French Guiana, and quarantine measures have been enforced to prevent the spread of this pathogen that might also cause severe losses on other susceptible plant species that are important for the local market (e.g., banana, coconut, sugar cane). Pineapple has become a major crop in French Guiana, and is now subjected to a more intensive monitoring, which may explains why this disease was discovered recently.

First Report of Diaporthe pseudomangiferae Causing Inflorescence Rot, Rachis Canker, and Flower Abortion of Mango.

Although mango (Mangifera indica L.) is a very important tropical fruit crop, limited studies have been conducted on fungal pathogens affecting the inflorescences. During a disease survey conducted from 2008 to 2010, 50% of the inflorescences were affected with inflorescence rot, rachis canker, and flower abortion characterized by blackening of plant tissue with soft rot lesions and suken lesions on the rachis, respectively. Symptoms were observed at the Mango Germplasm Collection of the University of Puerto Rico's Experiment Station in Juana Diaz, Puerto Rico. Five diseased pieces of 350 inflorescences from cvs. Haden and Irwin were disinfested with 70% ethanol, followed by 0.5% sodium hypochlorite, rinsed with sterile water, and transferred to acidified potato dextrose agar (APDA). Among several typical or common fungi, three isolates of Diaporthe pseudomangiferae (Dp) R.R. Gomes, C. Glienke & Crous were obtained from symptomatic tissue and identified morphologically using taxonomic keys and DNA sequence comparisons (1,2). On APDA, colonies of Dp initially had white-gray moderate aerial mycelia. Pycnidia were black and superficial on cultures with a central ostiole that exuded beige to light orange conidial droplets. Alpha conidia (n = 50) were aseptate, hyaline, smooth, fusiform, apex rounded and base truncate, averaged 7.34 μm long by 2.60 μm wide. Beta conidia (n = 50) were spindle-shaped, aseptate, hyaline and smooth, averaged 22.03 μm long by 1.53 μm wide. DNA analysis of the ITS1-5.8S-ITS2 region using primers ITS5 and ITS4, and fragments of both β-tubulin and translation elongation factor 1 alpha (EF1-α) genes using primers T1 and Bt2b, and EF1-728F and EF1-986R, respectively, were sequenced and compared using BLASTn with sequences available in the GenBank. Accession numbers of gene sequences of Dp submitted to GenBank were KF616498 to KF616500 for ITS region, KF616501 to KF616503 for β-tubulin, and KF616504 to KF616506 for EF1-α. For all genes used, sequences were 99 to 100% identical to reference isolate CBS 388.89 of Dp in GenBank. For each fungal isolate, pathogenicity tests were conducted on six random healthy non-detached mango inflorescences for both cvs. Haden and Irwin. Inflorescences were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on APDA and kept in a humid chamber using plastic bags for 8 days under field conditions. Untreated controls were inoculated with APDA disks only. The test was repeated twice. On cv. Haden, isolates of Dp caused rachis canker (sunken lesion on the rachis) at 8 days post inoculation (dpi). On cv. Irwin, isolates of Dp caused inflorescence rot. Initially, white mycelia was observed on inflorescences but eventually inflorescences turned brown and flower abortion was observed at 8 dpi. Untreated controls did not show any of the above symptoms and no fungi were re-isolated from tissue. From diseased inflorescences, Dp was re-isolated, thus fulfilling Koch's postulates. Diaporthe spp. have been associated with fruit rots, stem cankers, decay, and wilt on a wide range of plant hosts (3,4). Recently, Dp was associated with fruit peel of mango in Mexico and the Dominican Republic (2). To our knowledge, this is the first report of Dp causing inflorescence rot, rachis canker, and flower abortion in mango.

Fruit Rot of Tinda Caused by Pseudomonas aeruginosa-A New Report from India.

Fruit rot disease (FRD), an emerging problem of tinda (Praecitrullus fistulosus) in India. FRD epidemics begin during rainy and warm weather and often spoil marketable produce. Symptoms appear as numerous, pale brown-to-dark brown, deeply penetrating circular soft rot lesions on fleshy fruit tissues. Noneffervescent bacterial exudates occasionally form on lesions. Repeated isolations from FRD-affected tinda fruits consistently yielded the same bacterial species. Inoculation of the isolated bacterium into asymptomatic tinda fruits produced identical soft rot symptoms. Fruits were inoculated with the isolate ITCC B0030 (0.1 OD) by removing a 2.0-cm deep tissue plug with a sterile cork borer (5 mm in diameter) and injecting the inoculum with a syringe in the cylindrical cavity. After inoculation, the plug (upper 5 mm) was reinserted, sealed with sterile paraffin, and covered with a small piece of wet absorbent cotton to prevent dehydration. High humidity (>90%) and 30 to 33°C temperature was maintained after inoculation in a glasshouse. After 4 to 10 days, fruits showed FRD symptoms. The reisolated bacterium from artificially inoculated symptomatic fruits was identical with the original inoculated bacterium. Identity of the bacterial pathogen for FRD was confirmed by phenotypic and genotypic methods. The causal bacterium was a gram-negative, non-sporing motile rod with a single polar flagellum. The bacterium produced yellowish green and blue-green diffusible pigments on King's B (KB) medium. On yeast dextrose calcium carbonate agar at 30°C, the colonies produced abundant, blue, diffusible pigment within 48 h. The bacterium grew at temperatures up to 42°C but not at 4°C. Excellent growth occurred on Salmonella-Shigella agar and MaConkey's medium, as reported also for Pseudomonas aeruginosa strain P8. The bacterium produced ammonia, hydrogen sulfide, arginine dihydrolase, urease, lipase, catalase, gelatinase, and casinase but not amylase, indole, or acetyl methyl carbinol. The bacterium was identified as P. aeruginosa using Biolog based Bacterial Identification System version 4.2 (Biolog Inc., Hayward, CA). The bacterium did not utilize cellobiose, dulcitol, maltose, sorbitol, sucrose, arabinose, and starch. Upon infiltration on tobacco leaves (Nicotiana tabacum cv. Xanthi) at 107 or more cells ml-1, the bacterium gave a strong hypersensitive reaction within 24 h. Transmission electron micrographs (TEM, KYKY 1000B, Japan) of the causal bacterium revealed a single, polar flagellum. Identity was further confirmed as P. aeruginosa based on 16S rRNA sequence (1,491 nt) analysis with universal primers F1 (5'-GAGTTTGATCCTGGCTCAG-3') and R13 (5'-AGAAAGGAGGTGATCCAGCC-3'). A blastN search of GenBank revealed a >99% nt identity with P. aeruginosa strain TAUC 7 (HQ914782). The 16S rRNA gene sequence (1,491 nt) was deposited in Bankit GenBank (JF797204). To our knowledge, this is the first report of fruit rot of tinda caused by P. aeruginosa in India (ITCC B0030) and a new record of bacterial rot of Praecitrullus fistulosus induced by a fluorescent and blue-green pigment producing P. aeruginosa. To date, P. syringae pv. lachrymans and a nonfluorescent P. pseudoalcaligenes subsp. citrulli were reported to infect Citrullus lanata (1) and Praecitrullus fistulosus (2), respectively.

A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi

A medium was developed for the isolation and differentiation of Erwinia chrysanthemi from other Erwinia spp. based on the production of blue-pigmented indigoidine. The medium, named NGM, consists of nutrient agar supplemented with 1% glycerol, that induces pigment production, and 2 mM MnCl 2·4H 2O, that further enhances color development. More than fifty E. chrysanthemi strains from six different plant hosts were tested. All tested strains of E. chrysanthemi grew well on the NGM medium, developing dark brownish to blue colonies easily distinguishable from other Erwinia spp. The results indicate that pigment production on the NGM medium is a very stable property and can be used as a phenotypic property to differentiate E. chrysanthemi from other Erwinia spp. In addition, a specific oligonucleotide primer set was designed for the detection of indC, which is involved in indigoidine biosynthesis. All E. chrysanthemi strains tested contained indC as determined by PCR amplification. No amplification was observed with other Erwinia spp. Thus, pigment production of E. chrysanthemi on the NGM medium is consistent with the existence of indC. The NGM medium was used to isolate and identify the causal agent of soft rot lesions of diseased Phalaenopsis orchids from three orchid cultivation areas in Taiwan. The causal agents of Phalaenopsis soft rot were all identified as E. chrysanthemi. The results indicate that the NGM medium is efficient in isolation and identification of E. chrysanthemi from plants with soft rot symptoms and can also be used for epidemiological studies.

Reaction of arracacha genotypes to the root soft rot caused by Pectobacterium chrysanthemi

The purpose of this paper was to screen thirty-two arracacha genotypes for their reaction to root soft rot. Twenty roots of each genotype were inoculated with two Pectobacterium chrysanthemi isolates in a randomized experiment (10 roots/isolate). After inoculation, roots were individually wrapped with PVC film and kept at 26ºC in closed plastic bags. Soft rot lesions were recorded after 36 hours and genotypes were grouped in four classes of susceptibility by cluster analysis: 10 were less susceptible, 16 intermediate, 3 susceptible and 3 very susceptible. All the tested arracacha genotypes showed only variation in the degree of susceptibility.

Outbreak of Choanephora Blight Caused by Choanephora cucurbitarum on Green Bean and Pepper in Florida.

Severe outbreaks of Choanephora blight on green bean (Phaseolus vulgaris cvs. Bronco, Shade, and Gold Mine) and bell pepper (Capsicum annuum cvs. Aristotle, Crusader, and Sentry) were widespread in southwestern (Hendry and Collier counties) and northern Florida (Alachua County) in October and November 2002. Disease incidence, estimated by inspecting 100 randomly selected bean plants in each of four fields, was 40 to 100% and infected fruit ranged from less than 10 to 100%. Incidence estimated similarly on pepper plants in three fields was 35 to 40% with substantial fruit infection observed predominantly around the calyx. Zucchini fruit and a pigweed plant (Amaranthus sp.) were observed with sporulating lesions of Choanephora, indicating that other hosts were affected during the outbreak. Symptoms were blighted leaves, dieback of shoot tips, blighted blossoms, and black, soft-rot lesions on fruit. Choanephora sp. was sporulating in abundance on diseased tissue. Isolates of Choanephora sp. grew readily as pure cultures on acidified potato dextrose agar and malt yeast extract (MYE) agar. C. cucurbitarum (Berk. & Rav.) Thaxter was identified on the basis of shape and ornamentation of the sporangiola (1). The sporangiola of C. cucurbitarum are ellipsoid to broadly ellipsoid, and the wall is usually longitudinally striate. Pathogencity tests consisted of spray inoculation (5,000 spores per ml) of five 6-week-old plants each with and without wounding made by lightly scratching the leaf surface with a needle. Plants were placed in the greenhouse with temperatures ranging from 21 to 26°C, and symptom development was observed as early as 3 days after inoculation. The percentage of infected plants after wounding was 40% for bell pepper ('Enterprise'), 100% for green bean ('Opus'), 0% for watermelon (Citrullus lanatus 'Star Gazer'), 60% for cantaloupe (Cucumis melo 'Vienna'), and 20% for cucumber (Cucumis sativus 'Thunder CY'). Lesions on inoculated leaves were similar to those seen in the field on bean and pepper, and sporulation of C. cucurbitarum was present in the necrotic areas on all symptomatic plants. Pure cultures of C. cucurbitarum were reisolated. C. curcurbitarum was observed and isolated from a few noninoculated bean flowers and two noninoculated bean pods indicating spread to noninoculated plants; otherwise control plants were asymptomatic. Unwounded plants did not develop lesions, indicating that wounding was necessary for infection by this inoculation technique. The mating type was determined by juxtaposing several isolates on MYE agar, and zygospore formation was observed indicating both + and - strains occur in Florida. These outbreaks show that under the proper environmental conditions, such as long periods of high rainfall, high humidity, and high temperatures, crops like bean and pepper that are not usually affected by the disease may experience significant damage. Reference: (1) P. M. Kirk. Mycol. Pap. 152:1-61, 1984.

First Report of Foliar and Root Infection of Carrot by Sclerotinia minor in Ontario, Canada.

In 2000 and 2001, commercial carrots (Daucus carota L.) cv. Cellobunch grown in organic soils in Ontario, Canada, developed water-soaked, dark olive-green lesions on leaves that were in contact with soil. Lesions spread rapidly to petioles and adjacent leaves when prolonged moist conditions occurred within the canopy and persisted through harvest. A soft rot lesion was observed on the crown of one carrot root in the field, but no disease symptoms were detected on carrot samples in cold storage. Symptoms on leaves and roots of carrots were similar to sclerotinia rot caused by Sclerotinia sclerotiorum, a common disease in the area, except for the signs of sparser mycelia and smaller sclerotia. White mycelium and irregular, black sclerotia (0.5 to 2 mm) that formed on the surface of diseased leaf and root tissues were plated on potato dextrose agar (PDA) and incubated at 20 ± 1°C. Within 3 to 4 days, numerous mycelial clumps developed throughout the surface of white colonies and within 5 to 7 days these clumps developed into sclerotia. The pathogen was identified as S. minor Jagger (1). In the growth-room, young and senescing leaves of eight carrot (cv. Cellobunch) plants and 20 surface-disinfested roots were inoculated with 5-mm mycelial disks from the margins of colonies of one isolate of S. minor or S. sclerotiorum grown on PDA. A control treatment of an equal set of test units inoculated with sterile PDA plugs was also included. Inoculated plants and roots were incubated at 21 ± 1°C and ≈100% relative humidity for 21 days. Plants inoculated with S. minor, lesions, mycelial clumps, and sclerotia developed on the surface of leaves and petioles 1, 2, and 10 days postinoculation, respectively and were similar to those observed in the field. Lesions progressed faster on senescing leaves and reached the base of the petiole within 7 days, however, infection of the rosette or crown did not occur under these test conditions. In roots, lesions, mycelia, and sclerotia were observed 2, 4 and 10 days postinoculation, respectively, and sclerotia sometimes adhered in large aggregate crusts. S. minor was reisolated from infected tissues, completing Koch's postulates. Carrots inoculated with S. sclerotiorum yielded similar symptoms, but the white mycelium grew into cotton-like, dense mats and produced large (5 to 10 mm) individual sclerotia. Carrot has been previously reported as a host of S. minor (2), but to our knowledge, this is the first report demonstrating the occurrence of sclerotinia rot of carrot caused by S. minor in Ontario. S. minor may not cause damage to carrots in storage, but may affect yield by weakening the tops needed for efficient mechanical harvest. Carrot crops where S. minor was identified are typically rotated with onions (Allium cepa L.), which is not a host of S. minor (2). However, this 1-year rotation is unlikely to suffice for reducing the accumulation of sclerotial inoculum in soil. The knowledge of the occurrence and prevalence of S. minor in carrot crops is a valuable consideration when planning to incorporate carrots in rotation with other hosts of S. minor.

The adenylate cyclase (BAC) in Botrytis cinerea is required for full pathogenicity

SUMMARY The grey mould Botrytis cinerea is an economically important plant pathogen. Previously we found that null mutants of bcg1 encoding one of the two Galpha subunits of heterotrimeric GTP-binding proteins differed in colony morphology and showed reduced pathogenicity. To further understand the mechanisms involved in infection, we cloned the bac gene encoding adenylate cyclase, the enzyme that catalyses production of cAMP from ATP. The deduced protein sequence consists of 2300 amino acids, the ORF is interrupted by three conserved introns, and there is a high degree of similarity with the catalytic domains of other fungal adenylate cyclases. Gene replacement resulted in reduced vegetative growth and a morphology similar to that of bcg1 mutants. The wild-type (WT) colony morphology was partially restored by feeding exogenous cAMP. These bac mutants still had a low but constant level of cAMP, despite deletion of the complete catalytic domain of the enzyme. Conidia from bac mutants germinated, penetrated the leaves of Phaseolus vulgaris and caused spreading soft rot lesions (in contrast to bcg1 mutants), although these were slower to develop than in WT controls. Compared to the latter, the most striking difference was that no sporulation occurred on leaves inoculated with bac mutant conidia. These results confirm that the cAMP signalling pathway plays an important role in vegetative growth and pathogenicity in B. cinerea. On the other hand, a much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicity.

Effect of wet incubation time and temperature on infection, and of storage time and temperature on soft rot lesion expansion in potatoes inoculated withErwinia carotovora ssp.Carotovora

Infection and lesion expansion studies were carrried out on potato tubers. In the infection study, the tubers were inoculated withErwinia carotovora ssp.carotovora (Ecc) and exposed to incubation temperatures of 10–25 °C for 3–48 h of wetness. In the lesion expansion study, the tubers were exposed to 12 h of wetness at 20 °C to establish infection, and then·stored at temperatures of 4–16 °C for 15–90 d, at 95% RH. The volume of diseased tissue was determined and the data were transformed to proportion of maximum volume diseased (PVD). Infection was close to zero for 3 and 6 h wet incubation time, regardless of incubation temperature, and reached the maximum in 12 h of wetness at 20 °C. Significant lesion expansion occurred at a storage temperature of 16 °C after about 60 d of storage time. Cubic models of infection and lesion expansion potentials explained 95 and 96% of the variations in infection and lesion expansion, respectively.

Botrytis cinerea induces the formation of free radicals in fruits of Capsicum annuum at positions remote from the site of infection.

Free radical adducts of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits of Capsicum annuum investigated 5 days after infection with Botrytis cinerea. The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea.

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.