First report of collar and root rot of faba bean caused by Rhizoctonia solani AG-2-2 IIIB in Bangladesh

Faba bean (Vicia faba L.) is an underutilized promising grain legume commercially grown in central and northern part of Bangladesh (Yasmin et al. 2020). In January 2021, faba bean plants exhibiting symptoms of collar and root rot and yellowing of leaves were observed in thirty plots of an experimental field at the Bangladesh Agricultural University (24.75° N, 90.50° E), Mymensingh, Bangladesh. Infected plants had dark brown to black lesions on the roots, extending above the collar region. An average disease incidence and severity was 7.16% and 6.91%, respectively. Eight diseased plants were collected from the field by uprooting one plant from each of eight randomly selected experimental plots and surface disinfected with sodium hypochlorite (0.2%) for 3 min followed by 1 min in ethanol (70%), and then rinsed three times with distilled water and dried on sterile paper towels. Collar and root pieces (5×5 mm) of symptomatic tissues were placed on Potato Dextrose Agar (PDA). Plates were incubated at 25°C for three days and isolates were purified from single-tip culture. The isolates produced brown colored mycelia often with brown sclerotia. Under microscope, fungal colonies exhibited right–angled branching with constriction at the base of hyphal branches and a septum near the originating point of hyphal branch consistent with the description of Rhizoctonia solani Kuhn (Sneh et al. 1991). The isolates grew at 35°C on PDA (5 mm/24). Molecular identification of the isolates BTRFB1 and BTRFB7 was determined by sequencing the rDNA internal transcribed spacer (ITS) region using primers ITS1 and ITS4 (White et al. 1990). A BLAST search showed that the sequences (GenBank Accession nos. MZ158299.1 and MZ158298.1) had 99.28% similarity with R. solani isolates Y1063 and SX-RSD1 (GenBank Accession nos. JX913811.1 and KC413984.1, respectively). Phylogenetic analysis revealed that the present isolates grouped with R. solani anastomosis group AG-2-2 IIIB. To confirm pathogenicity, both isolates were grown individually on sterile wheat kernels at 28°C for 6 days (D’aes et al. 2011). Faba bean seedlings were grown in plastic pots containing sterile potting mix (field soil/composted manure/sand 2:2:1 [v/v]). Two-week-old plants were inoculated by placing five infested wheat seeds adjacent to the roots. Control pots were inoculated with sterile wheat kernels using the same procedure. Plants were placed in a growth room with a 16 h/8 h light/dark photoperiod at 25 ± 2°C after inoculation. Fifteen days after inoculation, typical collar and root rot symptoms were developed on inoculated plants, similar to symptoms observed in the field. Control plants remained non-symptomatic. Finally, six isolates of R. solani were isolated from the symptomatic plants and identified by morphological and molecular analysis. Rhizoctonia solani is the causal agent of seed and root rot, hypocotyl canker, and seedling damping-off diseases of faba bean in many other countries (Rashid and Bernier 1993; Assunção et al. 2011). To our knowledge, this is the first confirmed report of Rhizoctonia solani causing collar and root rot of faba bean in Bangladesh. This finding will be helpful for the development of management strategies to control this disease and to expand the production of faba bean in Bangladesh.

Phytophthora mediterranea sp. nov., a New Species Closely Related to Phytophthora cinnamomi from Nursery Plants of Myrtus communis in Italy

Monitoring surveys of Phytophthora related diseases in four forest nurseries in Italy revealed the occurrence of fourteen Phytophthora species to be associated with collar and root rot on fourteen plants typical of Mediterranean and alpine regions. In addition, a multilocus phylogeny analysis based on nuclear ITS and ß-tubulin and mitochondrial cox1 sequences, as well as micromorphological features, supported the description of a new species belonging to the phylogenetic clade 7c, Phytophthora mediterranea sp. nov. Phytophthora mediterranea was shown to be associated with collar and root rot symptoms on myrtle seedlings. Phylogenetically, P. mediterranea is closely related to P. cinnamomi but the two species differ in 87 nucleotides in the three studied DNA regions. Morphologically P. mediterranea can be easily distinguished from P. cinnamomi on the basis of its smaller sporangia, colony growth pattern and higher optimum and maximum temperature values. Data from the pathogenicity test showed that P. mediterranea has the potential to threaten the native Mediterranean maquis vegetation. Finally, the discovery of P. cinnamomi in alpine nurseries, confirms the progressive expansion of this species towards cold environments, probably driven by climate change.

Neomyzeten – eine anhaltende Bedrohung für den Schweizer Wald

Neomycetes – persistent threats to Swiss forests More than 40 neomycetes pathogenic to woody plant species were introduced to Switzerland during the last 100 years, and the number is increasing exponentially. A few neomycetes are invasive and cause serious diseases on forest and ornamental trees: Cryphonectria parasitica, causal agent of chestnut blight, Hymenoscyphus pseudoalbidus causing ash dieback, Ophiostoma novo-ulmi causing Dutch elm disease and Ceratocystis platani causing canker stain of plane. The causal agents of sudden oak death (Phytophthora ramorum) and collar and root rot of alder (P. alni ssp. alni) have been detected in Switzerland but have not reached epidemic levels. Ceratocystis fagacearum, causal agent of oak wilt, or Leptographium wageneri causing black stain root disease of conifers are absent from Switzerland, but are threats to Swiss forests and measures have been implemented to prevent their introduction. Prevention of threats by fungi which are harmless symbionts in their native range, e.g. endophytic fungi, is more challenging. The only way to avoid the endophyte problem is to grow trees from aseptic seed, saplings or meristem cultures. Additionally, setup of sentinel plantings in as many regions as possible all over the world could serve as early warning systems. To prevent the introduction of wilt and root diseases, only treated wood and treated potting media should be allowed for import. Natural regeneration prevents contamination of forest soils by Phytophthora species originating from nursery plants.

First Report of Collar and Root Rot of Physic Nut (Jatropha curcas) Caused by Neoscytalidium dimidiatum in Brazil

Physic nut (Jatropha curcas L.; Euphorbiaeae) has become important in Brazil due to its potential as a feedstock for biodiesel production. In October 2010, during routine monitoring of fields in the state of Piauí, several plants were observed with symptoms of collar and root rot. Initially, plants appeared withered and chlorotic, and later became defoliated. Roots and collars of affected plants also appeared rotten with black fungal structures directly observed. Symptomatic tissue fragments of 5 mm diameter were washed with 70% ethanol, 1% sodium hypochlorite, and sterilized water, before being placed in petri dishes containing potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were then obtained by single-spore isolation. The fungus isolated was grown on plates containing 2% water agar overlaid with sterilized corn straw or pine twigs and incubated at 25°C under a photoperiod of 12 h for 4 weeks to induce the formation of fruiting bodies. Thirty measurements of all of the relevant morphological characters were made using a light microscope for the identification of the species. On PDA, isolates initially appeared white and became dark after 7 days. The aerial mycelia formed chains of zero- to one-septate arthroconidia, oblong to globose, initially hyaline that became brown and with a thick wall with age. The dimensions of arthroconidia were 4 to 12 × 2.5 to 8 μm. The formation of pycnidia was observed on the plates with corn straw and pines twigs. These were dark, with a globose base up to 250 μm and a neck up to 810 μm. Conidiogenous cells were holoblastic, lageniform to ampulliform, hyaline, and 6 to 10 × 1.5 to 2.5 μm. Conidia were hyaline, ellipsoid to nearly fusiform, and 8 to 12 × 4 to 5 μm. Septate and dark conidia were not observed. DNA was extracted from one isolate following Wizard Genomic DNA Purification Kit procedures and amplified using primers ITS1 and ITS4. Products were directly sequencing by Macrogen, Korea. The 856-bp sequence obtained was deposited in GenBank (Accession No. JQ927342). The sequence was 99% similar to Neoscytalidium dimidiatum (Penz.) Crous & Slippers, further supporting the identification by morphology. Pathogenicity tests were conducted by using 6 mm disks removed from the outer bark of the collar region of healthy plants using a sterile cork borer, and 6-mm diameter plugs were placed in each wound. Five plants were inoculated with the isolate and five plants inoculated with an isolate-free agar plug. Below these, pieces of moistened cotton were placed and covered with Parafilm. After 60 days, all inoculated plants reproduced the symptoms observed in the field, and the pathogen was successfully reisolated. All non-inoculated plants remained healthy. The genus Neoscytalidium includes species having Scytalidium-like synanamorphs in the aerial mycelia and Fusicoccum-like conidia in the pycnidia (1). Currently, this species is reported to cause diseases in fig, mango, and orange (2,3). To our knowledge, this is the first report of collar and rot root caused by N. dimidiatum in J. curcas and of this fungus in Brazil. It seems likely the disease exists in areas beyond Piauí and could cause important losses for biodiesel production. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) G. Polizzi et al. Plant Dis. 93:1215, 2009. (3) J. D. Ray et al. Austral. Plant Dis. Notes 5:48, 2010.

First Report of Collar and Root Rot Caused by Phytophthora tentaculata on Witloof Chicory (Cichorium intybus) in Italy

Witloof chicory (Cichorium intybus L.) is an important crop in Italy where most of the crop is still produced in soil. In September 2009, chicory plants (cv. Pan di Zucchero) grown on a commercial farm in Tarquinia (central Italy) showed symptoms of a previously unknown disease. Symptoms, observed 20 days after transplanting, consisted of stunting, yellowing of leaves, and a crown and root rot. Affected plants turned brown, wilted, and eventually died. At the soil level, dark brown-to-black water-soaked lesions coalesced and often girdled the stem. All of the crown and root system was affected. At this location, the disease was severe and widespread, with 60% of observed plants being affected. A Phytophthora-like organism was consistently isolated on a medium selective for oomycetes (4) after disinfestation of lower stem and root pieces of C. intybus for 1 min in a solution containing 1% NaOCl. Tissue fragments of 1 mm2 were excised from the margins of the root and crown lesions. The pathogen genus was identified as Phytophthora based on morphological and physiological features. Sporangia were produced for identification by growing a pure culture for 15 days on modified V8 juice agar medium (Campbell V8 juice [200 ml], agar [15 g], CaCo3 [0.5 g], and sterile water [800 ml]) under alternating light and dark (12/12 h). Sporangia were pyriform to ovoid, papillate, and measured 33.3 to 59.2 × 18.9 to 30.2 μm (average 39.9 × 25.8 μm). Chlamydospores developed in 28-day-old cultures and measured 21.3 to 30.2 × 19.5 to 29.7 μm (average 24.4 × 23.6 μm). Oogonia were globose and measured 26 to 41 μm (average 32.5 μm). Eighty percent of antheridia were paragynous. Amphyginous antheridia (15 to 20%) were also observed. Oospores were scarcely produced and measured 24 to 32 μm in diameter. The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 851-bp segment showed 100% homology with the sequence of Phytophthora tentaculata. The nucleotide sequence has been assigned GenBank Accession No. GU949536. Pathogenicity of this isolate was confirmed by inoculating C. intybus cv. Pan di Zucchero plants 20 days after transplant. The same isolate was grown for 15 days on a mixture of 70:30 wheat/hemp kernels and then 5 g/liter of the inoculum was mixed into a substrate containing a mixture of blond and black peat (15:85 vol/vol), pH 5.5. Five plants per 2-liter pot were transplanted and four replicates were carried out. Twenty noninoculated plants represented the control treatment. The trial was repeated. Plants were kept in two growth chambers at two temperatures (20 and 25°C). Symptoms similar to those observed in the field developed 7 days after inoculation. Twenty days later, 100 and 40% of the plants were dead at 25 and 20°C, respectively. Control plants remained symptomless. P. tentaculata was consistently reisolated from symptomatic plants. To our knowledge, this is the first report of P. tentaculata on C. intybus in the world ( http://nt.ars-grin.gov/fungaldatabases/index.cfm ). P. tentaculata was recently reported on lavender in Spain (2) and oregano in Italy (3). The economic importance of this disease is relatively low on most commercial farms. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) L. Alvarez et al. Plant Dis. 90:523, 2006. (3) P. Martini et al. Plant Dis. 93:843, 2009. (4) H. Masago et al. Phytopathology 67:425, 1977.