First Report of Anthracnose of Malva sylvestris Caused by Colletotrichum trifolii in China

Common mallow (Malva sylvestris L.) is a perennial medicinal plant in the Malvaceae family, which is native to Asia, Europe, and North Africa. In July 2012, typical symptoms of anthracnose disease, with a disease incidence of ~70%, were observed on common mallow in the Medicinal Herb Garden of Shenyang Pharmaceutical University, Liaoning, China. The fungus mainly infected the stalks and leaves of M. sylvestris. Pinpoint, brownish lesions initially appeared at the flowering stage and the disease spread within the field. The lesions on stems gradually enlarged and became dark brown, elliptical, and slightly concave. Concurrently, acervuli and mucilaginous conidial masses of the pathogen appeared on lesions under moist conditions. Conidia were hyaline, one-celled, cylindrical with both ends rounded, and measured 10.0 to 12.5 × 2.5 to 4.0 μm (mean 11.3 × 3.3 μm). The fungus was isolated from symptomatic tissues. Small pieces from leaves and stems were surface disinfested with 70% ethanol and 1.5% sodium hypochlorite for 1 min, then rinsed three times with sterile distilled water, and cultured on potato dextrose agar (PDA) at 25°C. The colonies on PDA had initially white aerial mycelia, and later became greenish black with regularly whorled rings. To confirm Koch's postulates, five 3-month-old plants of M. sylvestris were inoculated with a conidial suspension (105 conidia/ml) prepared from PDA cultures incubated for 14 days. Five non-inoculated plants served as controls. The plants were maintained in the greenhouse at 22 to 25°C and about 75% relative humidity under natural daylight. Typical symptoms on inoculated plants were reproduced after ~10 to 14 days, whereas control plants remained asymptomatic. The pathogen was successfully recovered from symptomatic tissues and re-identified, completing Koch's postulates. The internal transcribed spacer (ITS) and large subunit -28S (LSU) region of rDNA was amplified with primers ITS1/ITS4 and NL1/NL4, respectively, and sequenced. Phylogenetic trees (ITS and LSU) that were obtained using MEGE3.1 with the neighbor-joining method showed that both of the isolates fall in the Colletotrichum trifolii clade. The representative sequences (ITS and LSU) were deposited in GenBank (Accession Nos. KJ155692 and KJ920935). The fungus isolated from symptomatic tissues was identified as C. trifolii on the basis of morphological, cultural characteristics, and sequence analysis (2). According to previous references, C. orbiculare and C. malvarum on Malvaceae were respectively described in America and Europe (1,3,4). However, the isolate from M. sylvestris significantly differed from those of C. orbiculare and C. malvarum in cultural characteristics and sequence analysis. In this paper, the results showed that M. sylvestris is a new host of C. trifolii. To our knowledge, this is the first report of mallow anthracnose caused by C. trifolii in China. References: (1) J. A. Bailey et al. Phytopathology 86:1076, 1996. (2) U. Damm et al. Fungal Divers. 61:29, 2013. (3) K. Hyde et al. Fungal Divers. 39:147, 2009. (4) L. Tosi et al. Plant Dis. 88:425, 2004.

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First Report of Leaf Spot Caused by Didymella americana on Cassia nomame in China

Plant Disease ◽

10.1094/pdis-08-21-1799-pdn ◽

2021 ◽

Author(s):

Weiming Sun ◽

Lina Feng ◽

Xiaolei Wen ◽

Bojia Han ◽

Danrun Xing ◽

...

Keyword(s):

Sequence Analysis ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Rrna Gene ◽

Pathogenicity Test ◽

Koch’S Postulates ◽

First Report ◽

Initial Symptoms ◽

Koch's Postulates

Cassia nomame (Sieb.) Kitagawa is an annual plant in the Leguminousae family. The aerial parts of C. nomame have been used as tonic and diuretic in Korea and Japan (Syed et al. 2019). A leaf spot was observed on the leaves of a 1-year-old C. nomame landrace in Changli County (39.42°N, 119.10°E), Qinhuangdao City, Hebei Province during August to October in 2018. In many fields (n≥3), the disease incidence over 80% in the middle and late stage of plant growth. Symptoms on leaves in one field began with many small, dark necrotic spot lesions. Later, the lesions spread to round-to-oval, slightly sunken in the center, and large necrotic patches with indefinite margins. Finally, lesions coalesced and resulted in defoliation. Lesions were occasionally observed on the pods. Symptoms on the pods were initially small, dark spots and then expanded to large necrotic patches with irregular edges. Symptomatic tissues (n=32) from pods and leaves were cut into 3 to 8 mm2 squares, surface disinfested with 75% ethanol for 10 s, rinsed with sterile distilled water, then placed on potato dextrose agar (PDA) at 28℃. After 3 days, ten isolates with consistent characteristics were obtained with a frequency 52.6%. The isolates on PDA were round, initially pale and had little aerial mycelium, gradually turned olive green and had dense wool-like dense aerial mycelia after 3 days. Conidia were hyaline, smooth, solitary, and elliptical. The conidia measured 5.4 to 8.2 μm × 2.5 to 3.8 μm (n=50), and has two oil bodies positioning at opposite poles. Pigmented chlamydospores were spherical or nearly pear-shaped, and solitary. Black fructifications (pycnidia) were produced profusely on PDA after subculture for 3 days. All the isolates were similar to Didymella sp. in morphology (Aveskamp et al. 2009). Choice three isolates YSGUO8 YSGGUO8-a and YSGGUO8-b to be further characterized by sequencing of the internal transcribed spacer (ITS), actin gene, and 28S large subunit of the nuclear rRNA gene (LSU) (Zhang et al. 2017). The sequences of three strains (MK836417 MZ484072 and MZ484073 for ITS, MK837604 MZ593675 and MZ593676 for actin, MK843781 MZ836208 and MZ836207 for LSU, respectively) showed 99% to 100% similarity with Didymella americana K-004 (KY070279 for ITS,KY070285 for LSU), Phoma americana CBS 256.65 (FJ426973 for ITS, FJ426871 for actin, MH870196 for LSU) and P. americana CBS 185.85 (FJ426972 for ITS, FJ426870 for actin, GU237990 for LSU) in GenBank. The fungi were identified as D. americana (formerly P. americana or Peyronellaea americana) on the basis of morphological characteristics and sequence analysis. A pathogenicity test was conducted with three times on 1-year-old C. nomame strain at the 4 to 6 compound leaf stage. Conidia were obtained from 7-day-old PDA cultures grown at 28℃ with a 12-h photoperiod. Koch’s postulates were fulfilled by spray inoculating ten healthy young plants with 106 conidia per milliliter of D. americana strain YSGUO8, and sterile water as the control. After inoculation, the plants were managed at 28℃, 60% relative humidity and a 12-h photoperiod. After 5 to 8 days, the inoculated leaves developed small and dark spots lesions similar to those observed on the leaves with initial symptoms in the field. The control leaves remained symptomless. The same fungi were re-isolated from infected leaves by morphology observation and sequence analysis, confirming Koch's postulates. D. americana has caused leaves spot on Table Beet in New York (Vaghefi et al. 2016). To our knowledge, this is the first report of D. americana causing leaf spot of C. nomame in China.

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First Report of Anthracnose of Sunflower Sprouts Caused by Colletotrichum acutatum in New Mexico

Plant Disease ◽

10.1094/pdis-08-12-0805-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 838-838

Author(s):

J. M. French ◽

J. J. Randall ◽

R. A. Stamler ◽

A. C. Segura ◽

N. P. Goldberg

Keyword(s):

New Mexico ◽

Sequence Analysis ◽

The United States ◽

Disease Diagnosis ◽

Colletotrichum Acutatum ◽

Economic Losses ◽

Distilled Water ◽

Koch’S Postulates ◽

First Report ◽

Koch's Postulates

In December 2011, edible sunflower sprouts (Helianthus annus) of two different commercially grown cultivars (Sungrown and Tiensvold) exhibiting stem and cotyledon lesions were submitted to the New Mexico State University Plant Clinic for disease diagnosis. The sample originated from an organic farm in Santa Fe County where the grower utilizes a small indoor growing facility. Stem lesions were elongate, reddish brown, and often constricted, resulting in stem girdling. Lesions on the cotyledons were dark brown with tan centers and round to irregular in shape. In some cases, the entire cotyledon was blighted. Fungal hyphae were observed on some lesions using a dissecting microscope. Colletotrichum acutatum was isolated from stem and cotyledon lesions when symptomatic tissue was plated on water agar. Conidia were fusiform ranging from 6.4 to 18.4 μm long and 2.1 to 5.1 μm wide and averaged 11.9 μm × 3.4 μm. Spores were measured from cream-colored colonies produced on acidified potato dextrose agar. PCR amplification and sequence analysis of 5.8S ribosomal DNA and internal transcribed spacers I and II was performed using primers ITS4 and ITS6 (2). An amplification product of approximately 600 base pairs in size was directly sequenced (GenBank Accession No. JX444690). A BLAST search of the NCBI total nucleotide collection revealed a 99% identity to multiple C. acutatum (syn: C. simmondsii) isolates. Four isolates were identified as C. acutatum based on morphological characteristics and DNA analysis. Koch's postulates were performed using four isolates of the pathogen and the two commercial sunflower cultivars (Sungrown and Tiensvold) originally submitted for disease analysis. Sunflower seeds were imbibed in distilled water for 24 h then sewn into peat plugs. Prior to seed germination, 5 ml of a C. acutatum spore solution (1 × 106/ml) from each isolate was applied to five peat plugs using an atomizer. Control plants were inoculated with distilled water and otherwise treated identically. Both sunflower cultivars were inoculated with each isolate of the pathogen and the test was replicated twice. The sewn peat plugs were incubated for 5 days at 21°C and 50% relative humidity. Symptoms similar to the original samples were present on 100% of the sprouts after 5 days. PCR and sequence analysis performed on cultures obtained from lesions showed a 100% match to the original New Mexico isolates fulfilling Koch's postulates. In an indoor organic facility, such as the one in NM, this disease has the potential to be very difficult to manage and the potential to infect a high percentage of the crop resulting in significant economic losses. To our knowledge, this is the second report of C. acutatum on sunflower sprouts in the United States (1) and the first report in New Mexico. References: (1) S. T. Koike et al. Plant Dis. 93:1351, 2009. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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First Report of Fusarium crookwellense Causing Tip Blight on Cones of Hop

Plant Disease ◽

10.1094/pdis.2001.85.11.1208a ◽

2001 ◽

Vol 85 (11) ◽

pp. 1208-1208 ◽

Cited By ~ 3

Author(s):

S. J. Pethybridge ◽

F. S. Hay ◽

C. R. Wilson ◽

L. J. Sherriff ◽

G. W. Leggett

Keyword(s):

Late Summer ◽

Personal Communication ◽

Distilled Water ◽

Conidial Suspension ◽

Koch’S Postulates ◽

Disease Symptoms ◽

First Report ◽

Fusarium Crookwellense ◽

Koch's Postulates ◽

Sydney Australia

Hop (Humulus lupulus L.) is grown primarily for the alpha and beta acids produced in the strobile (cone) and used for bittering beer. In late summer (March) 2001, necrotic lesions covering the tips of cones of cvs. Agate, Nugget, and Willamette at hop farms in Tasmania, Australia, were observed. The necrotic lesions encompassed the proximal tips and affected between 5 and 60% of the cone; however, all bracts in the whorl were always affected. Diseased cones were observed in all seven gardens included in the survey. The incidence of plants with cone tip blight in ‘Nugget’ ranged from 5 to 30% in three gardens, in ‘Agate’ ranged from 3 to 10% in three gardens, and in the only ‘Willamette’ garden 30% of cones were affected. Pieces of infected hop cones (N = 55) were surface-treated for 1 min in 2% sodium hypochlorite, placed on 2% water agar, and incubated at 22 ± 2°C. Fusarium crookwellense Burgess, Nelson, & Toussoun was isolated from 95% of the cones (1). F. crookwellense was identified on carnation leaf agar by L. Burgess, University of Sydney, Australia. Koch's postulates were fulfilled by inoculating detached mature hop cones of cvs. Nugget and Willamette (N = 20 for each cultivar) with an atomized conidial suspension (3.5 × 105 spores of a single F. crookwellense isolate per milliliter) until runoff and incubated at 20 ± 2°C in a sealed container on plastic mesh over tissue wetted with sterile distilled water. Symptoms first appeared 5 days after inoculation and were identical to those found in the field. No disease symptoms were observed on cones subjected only to sterile distilled water. The pathogen was reisolated from diseased tissue on inoculated cones, completing Koch's postulates. Similar disease symptoms on hop cones have been described in Oregon and were associated with infection by F. sambucinum and F. avenaceum (C. Ocamb, personal communication). To our knowledge, this is the first report of the infection of hop cones by F. crookwellense. Reference: (1) L. W. Burgess et al. Laboratory Manual for Fusarium Research, 3rd ed. University of Sydney, Australia, 1994.

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First Report of Anthracnose on Hymenocallis littoralis Caused by Colletotrichum siamense in China

Plant Disease ◽

10.1094/pdis-02-21-0405-pdn ◽

2021 ◽

Author(s):

Yong Huang ◽

Yue Qin zhang ◽

Han Hu ◽

Nai Feng

Keyword(s):

Its Region ◽

Leaf Spot Disease ◽

Data Sets ◽

Conidial Suspension ◽

Single Spore ◽

Koch’S Postulates ◽

First Report ◽

Medicinal Value ◽

Sterile Needle ◽

Koch's Postulates

Spider lily (Hymenocallis littoralis (Jacq.) Salisb.) is a widely cultivated horticultural plant worldwide and has ornamental and medicinal value. Spider lily plants were seriously affected by a leaf spot disease in the campus of Guangdong Ocean University and gardens in Zhanjiang city in February 2018 with an incidence of 30 to 100%. Affected leaves usually developed small circular purple spots, which enlarged to oval spots and large irregular spots. The spots were brown at the center, deep purple at the border and surrounded by a yellow halo. Diseased cultivars were collected in Zhanjiang city, Gangzhou city in Guangdong province and and Zhangping city in Fujian province. Symptomatic leaf samples were disinfested with 1% NaOCl, and cultured on sucrose agar (PSA) at 28 °C for one week. Ten single-spore isolates were recovered from PSA medium. Colonies developing on PSA were grayish white with a regular border. Conidia were straight, hyaline with rounded ends, 4.3 to 6.1×12.8 to 32.1μm (n = 50 conidia of each isolate). Fungal mycelia were hyaline, septate, and branched. Conidia were born on a long conidiogenous cell, appressoria were oval, 6.7 to 10.7 × 5.2 to 6.2 μm (n=50). The isolates were morphologically identified as Colletotrichum sp. (Weir et al. 2012). Tests of pathogenicity were performed according to Koch's postulates using three isolates. Fresh wounds were made with a sterile needle on the healthy surface of leaves of H. littoralis at the 4- to 6-leaf stage and each leaf was covered with a piece of cotton drenched with 200 μL of conidial suspension (106 conidia/ml) from each isolate. Control seedlings were inoculated identically except sterile water was used to drench the cotton. Inoculated plants were placed in a moisturizing light incubator at 25℃ and 80% humidity under a 12-h light/dark cycle for 20 days and examined daily to monitor disease symptom development. Small round brown spots were observed at the inoculation sites 3 days after the inoculation. The brown spots developed into large brown lesions 5 days after inoculation. There were no symptoms observed in the water-inoculated plants. A Colletotrichum spp. strain based on morphology was consistently reisolated from leaves lesions fulfilling Koch’s postulates. For molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CAL), Tublin (Tub) and Apmat loci of three isolates were amplified using primer pairs of ITS4/ITS5, CL1C/CL2C, T1/T2 and AM-F/AM-R (Sharma et al. 2015). A phylogenetic tree derived from a neighbor-joining analysis of a concatenated alignment of ITS, CAL, Tub and ApMAT sequences was created. The accession numbers of three isolates GZHLCG, ZJHLCG and FJHLCG used in this study were MW553083, MN540457, MN540458 for ITS, MW553087- MW553089 for CL, MW553090-MW553092 for Tub and MW553084-MW553086 for ApMAT. The sequences of the three isolates were aligned with related species of Colletotrichum (Sharma et al. 2015). Analyses based on concatenated data sets of four genes showed that the sequences had high levels of identity to those of the C. siamense strains. According to both morphological and sequence analyses, the H. littoralis pathogen was identified as C. siamense. There is a report of foliar diseases on H. littoralis caused by Colletotrichum sp. (Tan et al., 2009). To our knowledge, this is the first report of anthracnose on H. littoralis caused by C. siamense in China. Identification of the pathogen provide valuable information for diagnosis and controlling this disease in H. littoralis.

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First Report of Brown Leaf Spot of Rice (Oryza sativa L.) Caused by Bipolaris sorokiniana in Pakistan

Plant Disease ◽

10.1094/pdis-05-21-1097-pdn ◽

2021 ◽

Author(s):

Hafiz Muhammad Usman Aslam ◽

Nasir Ahmad Khan ◽

Syed Ismat Hussain ◽

Yasir Ali ◽

Muhammad Raheel ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Bipolaris Sorokiniana ◽

Infected Leaf ◽

Potato Dextrose Agar Medium ◽

Koch’S Postulates ◽

First Report ◽

Brown Leaf Spot ◽

Brown Leaf ◽

Koch's Postulates

Brown leaf spot of rice is one of the major seed-borne diseases and can diminish grain production up to 52% (Barnwal et al. 2013). In 2018, infected leaf samples showing the typical symptoms of brown spots were collected from the vicinity of the University of Agriculture, Faisalabad (31°26'10.3"N 73°03'35.1"E). The symptoms were brown-dark spots, with gray-light gray or brown centers surrounded by dark margins and with chlorotic halos and of oval or cylindrical shapes (5 to 9 mm in diameter). Disease incidence averaged 61% across the seven fields observed. Leaves were collected from the seven infected fields and symptomatic leaf tissues of 5 mm2 were excised from representative necrotic spots in each. These tissues were surface disinfected with 70% ethanol, rinsed with sterile distilled water (SDW), dried by blotting on paper, and placed on potato dextrose agar medium. For pathogen growth, the plates were placed at 25oC (±2oC) with a 12-hour photoperiod for 5 days. Five samples from each of the infected fields were taken for pathogen isolation and among them ten isolates were sub-cultured and purified by using the single spore method. The resulting fungal colonies were fluffy and ranged in color from grayish black/black to light brown. Fifteen conidia were measured that are olivaceous-brown to dark brown in color, elliptical to oblong with narrow (tapered) ends, with 3-10 septa and 35.6-65.4 µm in length x 13.1-25.7 µm in width. Conidiophores were yellowish-brown, geniculate, and solitary (Pratt 2003). For molecular studies, rDNA of the internal transcribed spacer (ITS) region, translation elongation factor (tef), RNA polymerase II second largest subunit (rpb2) and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified by using the primers ITS1F/ITS4R (White et al. 1990), EF1-983F/EF1-2218R (Rehner and Buckley 2005), 5F2/7CR (O’Donnell et al. 2007), and GPD1/GPD2 (Berbee et al. 1999) respectively. The sequence of all the amplified gene regions of one SUL-1 isolate was deposited into GenBank with accession numbers MN314844 (ITS), MN326866 (tef), MN990457 (rpb2) and MN990456 (gpd). BLASTn queries of the obtained sequences (ITS, tef, rpb2 and gpd) showed 99-100% homology with the corresponding nucleotide sequences of B. sorokiniana (GenBank accession nos. GU480767, MF490855, LT715652 and MK558818 respectively). To fulfill the Koch’s postulates, twenty rice plants (cv. Basmati-385) were sprayed at 2 to 3 leaf stages by using the two representative isolates with a spore suspension of 105 spores/ml. SDW was sprayed on ten control plants. The plants were covered with polyethylene bags to keep the moisture contents and incubated at 25oC (±2oC) for 7 days. After a week, same symptoms as those described above were observed. In the repeated experiment, B. sorokiniana was re-isolated from the infected rice leaves and confirmed morphologically; fulfill the Koch’s postulates. With grave worry, the other species of the genus Bipolaris (B. oryzae, and B. victoriae) have also been found to the cause brown leaf spot of rice (Motlagh and Kaviani 2008). To our knowledge, this is the first report of Bipolaris sorokiniana causing brown leaf spot of rice in Pakistan. Because rice is highly consumable grain in Pakistan, so the rapid spread of this disease in the rice farming areas is of a serious concern.

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First Report of Azalea Petal Blight Caused by Pestalotiopsis guepini in Argentina

Plant Disease ◽

10.1094/pdis.2000.84.1.100c ◽

2000 ◽

Vol 84 (1) ◽

pp. 100-100 ◽

Cited By ~ 6

Author(s):

M. C. Rivera ◽

E. R. Wright

Keyword(s):

Buenos Aires ◽

Morphological Characteristics ◽

Potato Dextrose Agar ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Koch’S Postulates ◽

First Report ◽

Pure Cultures ◽

Pathogenicity Testing ◽

Koch's Postulates

The most important azalea (Rhododendron spp.) growing area in Argentina is located in the outskirts of Buenos Aires. A disease of the azalea flower was detected during surveys conducted during September 1998. Irregular brown spots were uniformly distributed on petals and resulted in a flower blight that did not lead to abscission of petals. Pieces of infected petals were surface-sterilized for 1 min in 2% NaOCl, plated on potato dextrose agar, and incubated at 24 ± 2°C. Pure cultures were identified as Pestalotiopsis guepini (Desmaz.) Steyaert (synamorph P. guepini Desmaz.) based on morphological characteristics (1,2). Inoculation for pathogenicity testing was carried out by spraying a conidial suspension (1 × 106 conidia per ml) on plants with previously punctured petals. Inoculated plants with unwounded flowers, as well as noninoculated controls, were included. Plants were incubated in moist chambers at 24°C. Symptoms appeared on all punctured flowers within 4 to 5 days. Petals were blighted by 9 days after inoculation and were covered with black acervuli by 12 days after inoculation. Unwounded and noninoculated controls remained symptomless. The pathogen was reisolated from inoculated flowers, completing Koch's postulates. Pathogenicity of P. guepini on azalea leaves in Argentina was reported in 1991. This is the first report of P. guepini causing disease on azalea flowers in Argentina. References: (1) J. E. M. Mordue. CMI Descr. Pathog. Fungi Bact. No. 320, 1971. (2) B. C. Sutton. 1980. The Coelomycetes. Commonwealth Mycological Institute, Kew, England.

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Occurrence of Fusarium Wilt Caused by Fusarium oxysporum on Common Sage in Argentina

Plant Disease ◽

10.1094/pd-90-0833a ◽

2006 ◽

Vol 90 (6) ◽

pp. 833-833

Author(s):

S. A. Gaetán ◽

M. Madia

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Buenos Aires ◽

Disease Incidence ◽

Wilt Disease ◽

Buenos Aires Province ◽

Conidial Suspension ◽

Koch’S Postulates ◽

Koch's Postulates ◽

Open Fields

Common sage (Salvia officinalis L.) is being increasingly grown commercially in Argentina for its medicinal properties and as ornamental plants. Although the crop can be produced in greenhouses, most of the crop production is in open fields in Buenos Aires, Córdoba, and Santa Fe provinces. During the last 3 years, common sage has repeatedly shown decline symptoms in several production fields in the southern region of Buenos Aires Province. In the spring of 2004, a serious common sage wilt disease developed under field conditions resulting in as much as 15% loss of plants. The disease, affecting 10-month-old common sage plants, was observed in this region in two commercial fields located at Sierra de la Ventana. Affected plants appeared in irregular patches throughout the rows. Diseased plants exhibited symptoms of chlorosis, wilting, and death. Lower leaves on wilted plants showed gradual yellowing, apical necrosis, and premature defoliation. At advanced stages of the disease, irregular, brown, necrotic areas on the leaves occurred. The necroses on affected leaf parts occasionally expanded and coalesced to form large necrotic lesions that turned the entire leaf brown. Other symptoms included stunting, black streaking on stems, and rotting of roots. Longitudinal sections through stems and roots showed severely necrotic vascular tissue. Pieces taken from stems and roots of diseased plants were plated on potato dextrose agar after surface sterilization with 1% NaOCl for 3 min. The plates were incubated in the dark for 2 days and then kept under 12-h alternations of NUV light/dark for 8 days. On the basis of morphological and cultural characteristics, two fungal colonies were identified as Fusarium oxysporum Schlechtend.:Fr. (1) and used in the following studies. Pathogenicity tests were carried out on 4-month-old healthy common sage plants. Koch's postulates were completed for two isolates by dipping the roots of seedlings in a conidial suspension (2 × 105 conidia/ml) of a single-spore isolate for 25 min. Plants were repotted in a sterilized soil mix (soil/sand, 2:1). The experiment was conducted in a greenhouse at 23 to 25°C and 75% relative humidity with no supplemental light. Within 14 days, all inoculated plants showed typical Fusarium wilt symptoms similar to that observed in the field. Plants exhibited yellowing followed by wilting of foliage, rotting of roots, brown vascular discoloration, and then eventually collapsed. Four weeks postinoculation, 90% of the plants were dead. No symptoms were observed on control plants dipped only in distilled water. The fungus was successfully reisolated from the symptomatic plants, fulfilling Koch's postulates in all instances. F. oxysporum had been previously reported in 1995 to cause a wilt disease in clary sage (Salvia sclarea L.) fields in North Carolina; the disease was detected at seedling stage, reducing plant stand as much as 40 to 50% (2). In Argentina, the pathogen that caused wilt symptoms on common sage had also been observed in 2002 in greenhouses on the outskirts of Buenos Aires, although the disease incidence was low. At this time, the disease could become a limiting factor in common sage production and further information regarding this pathogen within the region is needed. To our knowledge, this is the first report of the occurrence of Fusarium wilt caused by F. oxysporum on commercial S. officinalis in open fields in Argentina. References: (1) P. E. Nelson et al. Fusarium species. An Illustrated Manual for Identification. Pennsylvania State University Press. University Park, PA, 1983. (2) V. P. Subbiah et al. Plant Dis. 80:1080, 1996.

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First Report of Alternaria alternata Causing Postharvest Decay on Apple Fruit During Cold Storage in Pennsylvania

Plant Disease ◽

10.1094/pdis-08-13-0817-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 690-690 ◽

Cited By ~ 1

Author(s):

W. M. Jurick ◽

L. P. Kou ◽

V. L. Gaskins ◽

Y. G. Luo

Keyword(s):

Cold Storage ◽

Alternaria Alternata ◽

The United States ◽

Apple Fruit ◽

Conidial Suspension ◽

Single Spore ◽

Koch’S Postulates ◽

First Report ◽

Postharvest Decay ◽

Koch's Postulates

Alternaria rot, caused by Alternaria alternata (Fr.) Keissl., occurs on apple fruit (Malus × domestica Borkh) worldwide and is not controlled with postharvest fungicides currently registered for apple in the United States (1). Initial infections can occur in the orchard prior to harvest, or during cold storage, and appear as small red dots located around lenticels (1). The symptoms appear on fruits within a 2 month period after placement into cold storage (3). In February 2013, ‘Nittany’ apple fruit with round, dark, dry, spongy lesions were collected from bins at commercial storage facility located in Pennsylvania. Symptomatic apples (n = 2 fruits) were placed on paper trays in an 80 count apple box and immediately transported to the laboratory. Fruit were rinsed with sterile water, and the lesions were superficially disinfected with 70% ethanol. The skin was removed with a sterile scalpel, and tissues underneath the lesion were cultured on potato dextrose agar (PDA) and incubated at 25°C with constant light. Two single-spore isolates were propagated on PDA, and permanent cultures were maintained on PDA slants and stored at 4°C in darkness. Colonies varied from light gray to olive green in color, produced abundant aerial hyphae, and had fluffy mycelial growth on PDA after 14 days. Both isolates were tentatively identified as Alternaria based on multicelled conidial morphology resembling “fragmentation grenades” that were medium brown in color, and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in chains on simple conidiophores (2). Conidia ranged from 15 to 60 μm (mean 25.5 μm) long and 10 to 25 μm (mean 13.6 μm) wide (n = 50) with 1 to 6 transverse and 0 to 1 longitudinal septa per spore. To identify both isolates to the species level, genomic DNA was extracted from mycelial plugs and gene specific primers (ALT-HIS3F/R) were used via conventional PCR to amplify a portion of the histone gene (357 bp) (Jurick II, unpublished). Amplicons were sequenced using the Sanger method, and the forward and reverse sequences of each amplicon were assembled into a consensus representing 2× coverage. A megaBLAST analysis revealed that the isolates were 99% identical to Alternaria alternata sequences in GenBank (Accession No. AF404617), which was previously identified to cause decay on stored apple fruit in South Africa. To prove pathogenicity, Koch's postulates were conducted using organic ‘Gala’ apples. The fruit were surface disinfested with soap and water and sprayed with 70% ethanol to runoff. Wounds, 3 mm deep, were done using a sterile nail and 50 μl of a conidial suspension (1 × 104 conidia/ml) was introduced into each wound per fruit. Fruit were then stored at 25°C in 80 count boxes on paper trays for 21 days. Water only was used as a control. Ten fruit were inoculated with each isolate or water only (control) and the experiment was repeated once. Symptoms of decay observed on inoculated were ‘Gala’ apple fruit were identical to the symptoms initially observed on ‘Nittany’ apples obtained from cold storage after 21 days. No symptoms developed on fruit in the controls. A. alternata was re-isolated 100% from apple inoculated with the fungus, completing Koch's postulates. A. alternata has been documented as a pre- and postharvest pathogen on Malus spp. (3). To our knowledge, this is the first report of postharvest decay caused by A. alternata on apple fruit during cold storage in Pennsylvania. References: (1) A. L. Biggs et al. Plant Dis. 77:976, 1993. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, the Netherlands, 2007. (3) R. S. Spotts. Pages 56-57 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinkle, eds. American Phytopathological Society, St. Paul, MN, 1990.

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Konjac anthracnose is caused by Colletotrichum gloeosporioides in Yunnan Province of China

Plant Disease ◽

10.1094/pdis-05-21-0945-pdn ◽

2021 ◽

Author(s):

Li Cheng

Keyword(s):

Dna Sequences ◽

Yunnan Province ◽

Disease Incidence ◽

Negative Control ◽

Economic Losses ◽

Conidial Suspension ◽

Sterile Water ◽

Koch’S Postulates ◽

Query Cover ◽

Koch's Postulates

Konjac (Amorphophallus konjac) is an economically important traditional crop in Fengqing County, Yunnan Province, China. Anthracnose symptoms were observed on this crop in June and July of 2020. The plants developed round, oval, or irregular leaf spots with brown edges and central taupe spots, and exhibited serious defoliation and tree weakness. Disease incidence in affected fields reached up to 35%. Anthracnose caused significant economic losses in konjac production and became a limiting factor of the konjac industry in Fengqing County. To date, no control measures of konjac anthracnose have been reported and tested in China. To determine the causal pathogen, symptomatic leaves were collected and cut into 5 mm2 pieces. The leaf peces were surface sterilized in 70% ethanol for 10 s, followed by treatment with 0.1% mercuric chloride for 3 min and three rinses in sterile distilled water. The tissue pieces were transferred onto potato dextrose agar (PDA) and incubated at 28°C. After 4 days of incubation, hyphal tips from leaf pieces were transferred to new PDA to generate pure cultures. The hyphae were initially white, and then became dark green; red-orange conidial masses were observed on the mycelium plate surface at a growth rate of 13.14 mm/day. The conidia, observed under 400× magnification, were colorless, long-oval to fusiform, one-celled, and 15.4 to 18.2 × 3.3 to 5.9 μm in size. To identify the isolate, the genomic DNA of the pathogen was extracted using the CTAB method. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and β-tubulin (TUB) gene were PCR amplified using the ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b primer pairs, respectively (Lee et al. 2020). BLASTn search of the obtained 536 bp ITS fragment (GenBank accession no. MT785772), 229 bp GAPDH sequence (MW187543), and 717 bp TUB sequence (MW187544) revealed a 99.44% to 99.63% sequence homology (100% query cover) with ITS (JQ005152, 99.44%), GADPH (JQ005239, 99.63%), and TUB (JQ005587, 99.60%) sequences of the C. gloeosporioides type strain CBS 112999, respectively. The highest homology with other Colletotrichum species was only 98.16%, including C. siamense, the causal agent of anthracnose in A. paeoniifolius and A. konjac (Prasad et al. 2017; Wu et al. 2020). To complete Koch’s postulates, leaves of 3-month-old konjac plants grown in the field were sprayed with a conidial suspension (106 spores/ml) of the isolate YNFQ-1 (sterile water was used as a negative control). Approximately 5 days after inoculation with YNFQ-1, symptoms similar to those in natural conditions appeared, whereas the negative control plants and fruits inoculated with the sterile water had no disease. The pathogen was re-isolated (strain YNFQ-1) from inoculated leaf tissues, and its identity was confirmed with both morphological and molecular (DNA sequences) tools, thus fulfilling Koch's postulates. The culture properties, morphological characteristics, and molecular identification confirmed the identity of the pathogen as C. gloeosporioides. There have been many reports about anthracnose of Amorphophallus; C. siamense causes anthracnose on A. paeoniifolius in India (Prasad et al. 2017) and A. konjac in Hubei, China (Wu et al. 2020), and C. gloeosporioides causes anthracnose of A. muelleri in Yunnan, China (Yang et al. 2020). To the best of our knowledge, this is the first report of C. gloeosporioides causing anthracnose on A. konjac in Fengqing County, China. The results are expected to have important implications in the diagnosis, control, and future research of anthracnose on A. konjac.

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First report of Colletotrichum truncatum causing anthracnose in cassava in Brazil

Plant Disease ◽

10.1094/pdis-03-21-0571-pdn ◽

2021 ◽

Author(s):

Stella de Castro Santos Machado ◽

Willie Anderson Santos Vieira ◽

Ingrid Gomes Duarte ◽

Ana Gabriele Amaral ◽

Josiene Silva Veloso ◽

...

Keyword(s):

Continuous Light ◽

Negative Control ◽

Conidial Suspension ◽

Sterile Water ◽

Manihot Esculenta Crantz ◽

Koch’S Postulates ◽

First Report ◽

Leaf Spots ◽

Average Size ◽

Koch's Postulates

Cassava (Manihot esculenta Crantz) presents significant economic importance in Brazil and other developing countries due to its use in human and animal feeding. In 2019, cassava plants sampled in Pará state (Brazil) presented necrotic and irregular leaf spots, characteristic symptoms of cassava anthracnose. About 90% of the plants were symptomatic, and disease severity was higher during months with high temperature and humidity. Fragments of symptomatic tissues were removed from the lesion transition area, surface disinfested (45 s in 70% ethanol, 1 min in 1% NaOCl, and rinsed twice in sterile water), and plated on potato dextrose agar. Cultures were incubated at 25 °C under continuous light for 7 days. Among the obtained isolates, seven presented grey felt-like mycelium with white sectors, reverse greyish, and hyaline, aseptate, smooth-walled, falcate conidia with average size 20.7—30.7 (26.1 ± 2.1) × 2.4—4.8 (3.5 ± 0.5) μm. Phenotypical features were similar to C. truncatum (Damm et al. 2019). The representative isolate UFT/Coll87 was chosen for further assays. The identity of the isolate was determined by maximum likelihood analysis using sequences of actin (ACT, GenBank accession number MT321653), β-tubulin (TUB2, MT856673) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, MT800857) partial regions. Colletotrichum isolate from cassava nested with C. truncatum isolates in a clade with 100% support, being confidently assigned to this species. Koch’s postulates were fulfilled to confirm the pathogenicity of UFT/Coll87. Inoculation was carried out in three cassava plants by spraying a conidial suspension (106 conida mL-1) on unwounded leaves (three leaves per plant). Plants sprayed with sterile water represented negative control. Inoculated plants were kept in a humid chamber for 48 h, 25 °C, and a 12-h photoperiod. The experiment was repeated 2 times. Typical cassava anthracnose symptoms were observed 10 days after inoculation. No symptoms were observed in negative control. The pathogen was reisolated from symptomatic leaves and was phenotypically identical to the original isolate UFT/Coll87, fulfilling Koch’s postulates. Colletotrichum fructicola, C. karstii, C. plurivorum, and C. siamense were reported causing cassava anthracnose in China (Liu et al. 2019). In Brazil, C. chrysophilum, C. fructicola, C. siamense and C. theobromicola were reported in association with cassava (Bragança et al. 2016; Oliveira et al. 2018; Machado et al. 2020). To our knowledge, this is the first report of C. truncatum causing cassava anthracnose worldwide. Our finding is important for disease management due to the high host range of C. truncatum. The pathogen can reduce the cassava yield, and the crop may serve as a potential inoculum source since it is commonly cultivated near to other crops that are also infected by C. truncatum.

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