First Report of Black Spots on Avocado Fruit Caused by Neofusicoccum parvum in Mexico

Avocado (Persea americana L.) production for export markets has increased in Mexico during the past 10 years. The production system, however, is affected by several sanitation factors, including diseases. During the spring of 2009, smooth, black, circular spots were noted on the surface of avocado fruit. A study was conducted during the winter of 2010 to ascertain the etiology and identify the fungus associated with black spot symptoms on avocado fruit in orchards of Nuevo Parangaricutiro County (19°25′00″ and 102°07′43″) in Michoacan, Mexico. Several fungal isolates were obtained on potato dextrose agar (PDA) from the margin of lesions on immature fruit. The internal transcribed spacer region (ITS) of the rDNA from representative isolates was sequenced with universal primers ITS5 and ITS4 (2). BLAST searches in GenBank showed 100% similarity of the nucleotide sequences with Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, GenBank Accession Nos. GU188001 to GU188007 and GU187985 to GU187987. A representative nucleotide sequence of this region was deposited in GenBank under the Accession No. JN203129. Strains of N. parvum produced aerial and compact mycelium on acidified PDA, the anamorph state of Botryosphaeria parva. Mycelium was initially white, turning gradually gray to black. Conidia were one celled, hyaline, ellipsoidal to fusiform, externally smooth, thin walled, nonseptate, with one or two septa with age, and an average length and width of 14.5 (9.5 to 19) × 5.8 (4.0 to 7.2) μm (n = 100). Pathogenicity tests were conducted with six avocado fruit cv. Hass. Fruit were inoculated at three evenly spaced locations on the fruit surface, either by wounding the tissue with a needle that had been dipped in a conidial mass from an 8-day-old monoconidial culture of N. parvum strain CIAD-021-11 or by placing 5 μl of 1 × 106 conidia ml–1 suspension on each inoculation site. Inoculated fruit were maintained in a moist chamber at 25°C for 2 weeks. Black lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites 4 dpi. The delay of symptom development was likely due to penetration through the lenticels, which took longer to initiate infection. No symptoms were observed in the control fruit. The pathogen was reisolated from the lesions of all inoculated fruit, thus fulfilling Koch's postulates. The results confirmed the pathogenic potential of this fungus and indicated its possible involvement in the etiology of black spot on avocado fruit. N. parvum is a cosmopolitan, plurivorous pathogen causing disease in several hosts of economic importance, such as grapes and kiwi, as well as causing stem-end rot of avocado fruit in New Zealand (1) and avocado twigs in Spain (3). To our knowledge, this is the first report of N. parvum causing black spots on avocado fruit in Mexico. References: (1) W. F. T. Hartill et al. N.Z.J. Crop Hortic. Sci. 30:249. 2002. (2) T. J. White et al. Page: 315 in: PCR Protocols: A Guide to Methods and Application. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990. (3) T. Zea-Bonilla et al. Plant Dis. 91:1052, 2007.

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First Report of a Fruit Rot Disease of Avocado Caused by Neofusicoccum mangiferae

Plant Disease ◽

10.1094/pdis-93-7-0760b ◽

2009 ◽

Vol 93 (7) ◽

pp. 760-760 ◽

Cited By ~ 3

Author(s):

H. F. Ni ◽

R. F. Liou ◽

T. H. Hung ◽

R. S. Chen ◽

H. R. Yang

Keyword(s):

Average Length ◽

Persea Americana ◽

Plant Diseases ◽

Fruit Rot ◽

Postharvest Disease ◽

Fluorescent Light ◽

First Report ◽

Avocado Fruit ◽

Rot Disease ◽

Serious Disease

Production of avocado (Persea americana) has increased significantly during the last 10 years in Taiwan and the area of cultivation is approximately 500 ha. The most important postharvest disease of avocado is anthracnose caused by Colletotrichum gloeosporioides (Penz.) in Taiwan (1). In 2008, a new disease was found to be infecting avocado fruit at some orchards in Tainan County of southern Taiwan. Infected avocados developed smooth, brown, circular spots first on the surface of harvested fruits. A fungus was always isolated from the margin of lesions and could also be found from symptomless fruit pedicles and stems. Fungal colonies cultured on acidified potato dextrose agar (PDA with lactic acid; pH 3.8) were initially colorless, turned dark gradually, and ultimately became gray to dark gray. After 4 days under fluorescent light at 25°C, pycnidia formed on PDA. Conidia obtained from fruiting bodies were ovate, one celled, and hyaline, with an average length and width of 12.9 (9.9 to 15.6) × 6.4 (5.2 to 7.2) μm. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (No. EU847427). It showed a sequence identity of 99% with Neofusicoccum mangiferae ((Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips) (GenBank No. AY615185). Thus, both morphological and molecular results confirmed the isolated fungus as N. mangiferae. Five avocado fruits were used to test the pathogenicity with three different treatment inoculation sites on each fruit. Wounded and unwounded sites on fruit were inoculated with mycelia agar plugs (0.5 mm in diameter) excised from a monoconidial culture and the fruit was kept in a plastic box with high humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites at 4 dpi. The pathogen was reisolated from the lesions of inoculated fruits and found to be N. mangiferae, thus fulfilling Koch's postulates. In control experiments, sterile agar plugs were placed on the wounded avocado fruits. These fruits remained completely free from symptoms throughout the experiment. Several species of Botryosphaeria have been reported on avocado, including N. parvum (anamorph of B. parva), Fusicoccum aesculi (anamorph of B. dothidea), and Dothiorella aromatica (= F. luteum). To our knowledge, this is the first report of N. mangiferae causing fruit rot of avocado in Taiwan. Previously, N. mangiferae has been reported on mango trees worldwide, especially in Australia and Thailand (2). The presence of N. mangiferae in the subtropical area presents a serious disease problem not only to avocado but also to mango. References: (1) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002. (2) B. Slippers et al. Mycologia 97:99, 2005.

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Characterization and Pathogenicity of Botryosphaeriaceae Species Obtained from Avocado Trees with Branch Canker and Dieback and from Avocado Fruit with Stem End Rot in Chile

Plant Disease ◽

10.1094/pdis-07-18-1131-re ◽

2019 ◽

Vol 103 (5) ◽

pp. 996-1005 ◽

Cited By ~ 8

Author(s):

Ana L. Valencia ◽

Pilar M. Gil ◽

Bernardo A. Latorre ◽

I. Marlene Rosales

Keyword(s):

Vascular System ◽

Persea Americana ◽

Effective Control ◽

Future Research ◽

Severe Drought ◽

Internal Transcribed Spacer Region ◽

Avocado Fruit ◽

Hass Avocado ◽

Botryosphaeriaceae Species ◽

Branch Canker

Several species of the Botryosphaeriaceae family have been associated with branch canker, dieback, and stem end rot in avocado (Persea americana Mill.). In Chile, the incidence of diseases affecting the avocado tree increased from 2011 to 2016, which coincided with a severe drought that affected avocado production. Moreover, distant countries importing avocados from Chile also reported an increase of stem end rot of ripe avocados. Therefore, the aims of this study were to identify the pathogen species associated with branch canker, dieback, and stem end rot of avocado in Chile and to study their pathogenicity. This study was conducted between 2015 and 2016 in ‘Hass’ avocado orchards located in the main avocado-producing regions in Chile. A diverse collection of fungal species was recovered from both necrotic woody tissue and necrotic tissue on harvested ripe fruit. On the basis of morphology and phylogenetic analyses of the internal transcribed spacer region (ITS1-5.8S-ITS2) and the translation elongation factor 1-α (TEF1-α) gene, eight species in the Botryosphaeriaceae family were identified: Diplodia mutila, D. pseudoseriata, D. seriata, Dothiorella iberica, Lasiodiplodia theobromae, Neofusicoccum australe, N. nonquaesitum, and N. parvum. For each of these species, pathogenicity studies were conducted on 1-year-old healthy Hass avocado plants. All isolates produced brown gum exudate and caused necrosis in the vascular system 3 weeks after inoculation. N. nonquaesitum, N. parvum, and D. pseudoseriata were the most virulent species. Necrotic lesions and cavities with white mycelia near the peduncle union were observed on Hass avocado fruit inoculated postharvest. L. theobromae, N. australe, and N. parvum were significantly more virulent than the other tested species in the Botryosphaeriaceae family. This study identified and characterized the pathogenicity of Botryosphaeriaceae species in Chile, which will prove useful to future research on these pathogens directed at establishing effective control strategies in avocado.

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First Report of Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Croatia

Plant Disease ◽

10.1094/pdis-03-13-0283-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1656-1656 ◽

Cited By ~ 6

Author(s):

J. Kaliternam ◽

T. Milicevic ◽

D. Bencic ◽

B. Duralija

Keyword(s):

Aerial Mycelium ◽

Morphological Data ◽

Width Ratio ◽

Vitis Vinifera L ◽

Internal Transcribed Spacer Region ◽

Foeniculum Vulgare ◽

Neofusicoccum Parvum ◽

First Report ◽

Grapevine Trunk Diseases ◽

Trunk Diseases

In September 2010, during survey of diseased grapevines (Vitis vinifera L.) in vineyards at localities Zmajevac (BZ), Orahovica (SO), Cilipi (KC), and Novalja (PN), symptoms characteristic of grapevine trunk diseases (GTD) (3) were observed, showing on cross-sectioned cordons and trunks as brown, wedge-shaped perennial cankers and/or dark streaking of the wood. In Croatia, these symptoms were traditionally associated with Eutypa Tul. & C.Tul. and with fungi from Diaporthaceae (2). From affected grapevines (cvs. Grasevina, Pinot bijeli, Malvazija dubrovacka, and Gegic), samples of symptomatic cordons and trunks were collected (n ≥ 35). To isolate the causal agents from the samples, woodchips of symptomatic tissue, surface-sterilized in 2% sodium hypochlorite for 2 min, were placed on potato dextrose agar amended with streptomycin sulphate (50 μg/ml) and incubated for 7 days at 25°C in darkness. A percentage of samples (72, 15, 27, and 54% from BZ, SO, KC, and PN, respectively) yielded fungal colonies with abundant aerial mycelium, initially white, but turning olivaceous grey after 5 days. From these colonies, monohyphal isolates were obtained and pycnidial formation stimulated by cultivation on 2% water agar with stems of plant species Foeniculum vulgare Mill. at 25°C under diffuse light for 3 weeks. Pycnidia contained conidia that were hyaline, unicellular, ellipsoid with round apices and truncated bases, and thin walled with smooth surface. Dimensions of conidia (n ≥ 50) were (12.8) 15.3 ± 1.4 (17.6) × (5.4) 6.3 ± 0.8 (7.6) μm, with length/width ratio (2.0) 2.5 ± 0.5 (3.2). Based on morphological data, species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was suspected (1). For molecular identification, isolates BZ330, SO334, KC342, and PN121 were used for PCR to amplify internal transcribed spacer region and partial translation elongation factor 1-alpha gene, using primers ITS5/ITS4 and EF1-728F/EF1-986R, respectively. Obtained sequences were shown to be identical between the four isolates (GenBank: KF296318, KF296319) and when compared with sequences for reference N. parvum isolate CMW9080 (AY236942, AY236887) they showed >99% homology, confirming the isolates as species N. parvum. Pathogenicity tests were done by inoculation of detached green shoots (GS) and lignified canes (LC) (n = 5) of grapevine cv. Skrlet by either mycelial plugs of the same four isolates, or sterile agar plugs for the controls. Inoculated GS were kept in flasks with sterile water in a glasshouse for 10 days, and LC in humid dark chambers for 30 days, at 25°C. Resulting vascular necrosis measured 62 to 81 mm (GS) and 215 to 246 mm (LC), but was absent on controls. Koch's postulates were satisfied by successful reisolation of N. parvum only from plants inoculated with mycelial plugs. N. parvum has been recognized as a serious grapevine pathogen, causing similar symptoms worldwide (3). To our knowledge, this is the first report of N. parvum associated with GTD in Croatia, and due to its relatively high incidence at surveyed localities, it could present considerable threat, particularly for neighboring vine growing regions. Diplodia seriata De Not., a weak pathogen (3), was also identified from a percentage of samples in this survey. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) J. Kaliterna et al. Arh. Hig. Rada Toksikol. 63:471, 2012. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50(Suppl.):S5, 2011.

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First Report of Cercospora apii, Causal Agent of Cercospora Early Blight of Celery, in Serbia

Plant Disease ◽

10.1094/pdis-02-14-0135-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1157-1157 ◽

Cited By ~ 1

Author(s):

A. Milosavljević ◽

E. Pfaf-Dolovac ◽

M. Mitrović ◽

J. Jović ◽

I. Toševski ◽

...

Keyword(s):

Early Blight ◽

Molecular Data ◽

Causal Agent ◽

Apium Graveolens ◽

Universal Primers ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Molecular Features ◽

Sterile Needle ◽

Histone H3 Gene

Celery (Apium graveolens var. dulce) is a very important vegetable crop intensively cultivated in eastern and southern Serbia. During a field survey in August and September 2012, we observed symptoms similar to those of Cercospora early blight in eastern Serbia, with some of the affected fields showing up to 80% disease severity. The lesions on leaves were amphigenous, subcircular to angular and more or less confluent. Lesions enlarged and merged with age, followed by the development of necrotic area causing a continuous deterioration of the plant. Conidiophores arising from the stromata formed dense fascicles, sometimes appearing solitary, brown at the base, paler toward the apex, simple, straight to slightly curved, and rarely geniculate (dimensions 40 to 90 × 5 to 8 μm). Conidia were solitary, hyaline, at first cylindro-obclavate then acicular to acicular-obclavate, straight to slightly curved, subacute to obtuse at the apex, while truncated and thickened at the base (dimensions 45 to 160 × 4 to 5 μm), 5 to 13 septate. Based on the morphological features, we identified the pathogen as Cercospora apii Fresen. (2). In order to obtain monosporic isolates of the fungus, single conidia were cultivated on potato dextrose agar (PDA). To confirm the pathogenicity of the isolates, 5 mm-diameter mycelial plugs from the PDA plates were placed upside down on the adaxial leaf surface of 2-week-old celery seedlings of cv. Yuta. Control plants were inoculated with a sterile PDA plug. Three leaves per plant were disinfected with 70% ethanol, epidermis was scratched with a sterile needle to promote the infection, and inoculated. A total of 12 plants were inoculated with the mycelial plugs and 12 were used as control plants. Inoculated and control plants were kept in a moist chamber for 48 h and then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, the first necrotic spots appeared on inoculated leaves, similar to the symptoms manifested in the field, while control plants remained symptomless. The pathogen was re-isolated and its identity was verified based on morphological and molecular features. To confirm the pathogen's identity, three isolates (CAC4-1, CAC24, and CAC30) were subjected to molecular identification based on the internal transcribed spacer region (ITS) using the ITS1/ITS4 universal primers (5), a partial calmodulin gene (CAL) using CAL-228F/CAL2Rd primers (1,4), and partial histone H3 gene (H3) using CYLH3F/CYLH3R primers (3). Sequences of the amplified regions were deposited in GenBank under accessions KJ210596 to KJ210604. The BLAST analyses of the ITS sequences revealed 100% identity with several Cercospora species (e.g., C. apii [JX143532], C. beticola [JX143556], and C. zebrina [KC172066]), while sequences of CAL and H3 showed 100% identity solely with sequences of C. apii (JX142794 and JX142548). Based on combined morphological and molecular data, the pathogen infecting celery was identified as C. apii, which to our knowledge represents the first report of the presence of the causal agent of Cercospora early blight disease in Serbia. References: (1) I. Carbone and L.M. Kohn. Mycologia 91:553, 1999. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) J. Z. Groenewald. Stud. Mycol. 75:115, 2013. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

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First report of black spot on Ophiopogon japonicus caused by Alternaria alternata in Zhejiang Province, China

Plant Disease ◽

10.1094/pdis-06-21-1188-pdn ◽

2021 ◽

Author(s):

Yiwen Xu ◽

Zhenyan Cao ◽

Yihua Yang ◽

Jintian Tang ◽

Yang Song ◽

...

Keyword(s):

Alternaria Alternata ◽

Early Stage ◽

Black Spot ◽

Zhejiang Province ◽

Chemical Components ◽

Conidial Suspension ◽

Sterile Water ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Ophiopogon Japonicus

Ophiopogon japonicus (Linn. f.) Ker-Gawl, a traditional Chinese medicinal plant, is widely cultured in China. The root of O. japonicus, is used as the main ingredient in many presriptions. It is rich in chemical components for steroidal saponins, homoisoflavonoids and polysaccharides, which have various pharmacological activities, such as cardiovascular protection, anti-inflammation and anti-diabetes (Chen. et al. 2016). In May and July for 2018 and 2019, the symptoms of black spot on O. japonicus were observed with an incidence of 40% in Cixi County, Zhejiang Province, China. The pathogen mainly infected leaves causing severe black spots, which resulted in a 28% yield loss per acre. At the early stage of the disease, the tip of the leaf began to turn yellow, then the discoloration gradually spread to the base of the leaf and finally the whole leaf turned reddish brown with visible black spot. Symptomatic leaves were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30s and 1% NaClO for 30s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA), and incubated at 28 ℃ in dark for a week. Then, pathogen on the PDA were transferred onto potato carrot agar (PCA), and incubated at 23 ℃ under the condition of alternation of day (12 h) and night (12 h) for a week. Colonies on PDA were dark gray in the center surrounded by white to gray on the upper side, and black with white margins on the back of the plate. Colonies on PCA were grayish with sparse hyphae. The conidia were obclavate or ellipsoid, pale brown, with 3~8 transverse septa and 1~4 longitudinal septa. Conidiophores were septate, arising singly, and measured (17.0~81.0) × (8.0~23.5) μm, Most conidia had a conical or columnar beak, approximately (0~23.5) × (2.5~9.0) μm in size. According to morphological and cultural characteristics, these isolates were preliminarily identified as Alternaria alternata. A. alternata is one of the most typical plant pathogen, more than 95% of which facultatively parasitize on plants, causing disease in numerous crops. To further confirm identification of pathogens, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF-1α), RNA polymerase Ⅱ second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), Histon 3 gene (His) and plasma membrane ATPase (ATP)were amplified with primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-7cr/RPB2-5f2, Alt-for/Alt-rev, His 3-F/His 3-R, ATP-F/ATP-R (Lawrence D.P. et al. 2013; Hong, S.G., et al. 2005). BLASTN analysis of NCBI using ITS (Accession NO. MW989987), Alt a1 (Accession NO. MW995953), EF-1α (Accession NO.MW995955), ATP (Accession NO.MW995957), His (Accession NO. MW995954) and RPB2 (Accession NO. MW995956) showed 100%, 100%, 97%, 99%, 99% and 97% identity to A. alternata MN249500.1, MN304714.1, MK637432.1, MK804115.1, MK460236.1, MK605888.1, respectively. To verify pathogenicity, healthy plants (1-year-old) of O. japonicus in ten pots were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70–80% relative humidity and a photoperiod of 16:8 [L: D] h). Fourteen days later, all inoculated plants showed typical symptoms of black spot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as A. alternata by morphology observation and sequence analysis. To our knowledge, this is the first report of black spot caused by A. alternata on O. japonicus in Zhejiang, China.

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First Report of Neofusicoccum parvum Associated with Dieback of Mango Trees in Peru

Plant Disease ◽

10.1094/pdis-93-4-0426b ◽

2009 ◽

Vol 93 (4) ◽

pp. 426-426 ◽

Cited By ~ 12

Author(s):

J. Javier-Alva ◽

D. Gramaje ◽

L. A. Alvarez ◽

J. Armengol

Keyword(s):

Dna Sequences ◽

Mangifera Indica ◽

Elongation Factor ◽

Tree Canopy ◽

Internal Transcribed Spacer Region ◽

Neofusicoccum Parvum ◽

First Report ◽

Early Maturation ◽

Mycelial Plug ◽

Stem Lesions

Mango (Mangifera indica L) is one of the most important cash crops of northern Peru. Since 2003, adult mango trees (cvs. Criollo and Kent) located in Piura Province developed symptoms of dieback characterized by the death of twigs and branches in the tree canopy. Additional disease symptoms involved darkened, elongated lesions on the peduncle, causing an early maturation of the fruit, and in advanced symptoms, stem-end rot of fruits. Symptoms were frequent in the spring months (September to November) when the lesions expand rapidly. Diseased tissues from branches and fruits were collected and small pieces of necrotic tissues were surface disinfected and plated onto potato dextrose agar (PDA) with 0.5 g L–1 streptomycin sulfate. Plates were incubated at 25°C in the dark. All affected tissues consistently developed colonies with a white mycelium, moderately dense, and becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile mango twigs placed on the surface of potato carrot agar (PCA) after 10 days. Conidia were hyaline, guttulate, aseptate, measuring (15-) 18.5 (-22.5) × (4-) 5.2 (-7.5) μm. Conidia became olivaceous and developed one or two septa before germination. Isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, & A.J.L. Phillips (1). DNA sequences of the rDNA internal transcribed spacer region (ITS) and part of the translation elongation factor 1-alpha (EF1-α) genes were used to confirm the identification through BLAST searches in GenBank (ITS: 99% identity to Accession No. EU080928; EF1-α: 98% identity to Accession No. AY343367). Representative sequences of the studied DNA regions were deposited at GenBank (ITS: Accession No. FJ528596; EF1-α: Accession No. FJ528597). Pathogenicity tests were conducted on 18-month-old potted mango plants cv. Kent with two N. parvum strains (A4 and A5). A mycelial plug (3 cm in diameter) taken from the margin of an actively growing colony of each isolate was put in a wound made with a cork borer of the same diameter on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate were used with an equal number of control plants. Plants were maintained in a greenhouse with a temperature range of 22 to 28°C. After 4 weeks, mango plants showed necrotic stem lesions originating from the inoculation point affecting also the branches of the inoculated plants. No differences in lesion area between strains were obtained. No lesions developed in the control plants. Reisolations from necrotic tissues were successful and both isolates were morphologically identical to those used for inoculations. N. parvum was isolated from all symptomatic trees in all surveyed areas. This pathogen has already been reported on mango (2) and currently represents a serious problem in the mango-producing areas of Peru. To our knowledge, this is the first report of N. parvum affecting mango in Peru. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 97:99, 2005.

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First Report of Gray Leaf Spot on Pepper Caused by Stemphylium solani in Malaysia

Plant Disease ◽

10.1094/pdis-03-12-0262-pdn ◽

2012 ◽

Vol 96 (8) ◽

pp. 1227-1227 ◽

Cited By ~ 4

Author(s):

A. Nasehi ◽

J. B. Kadir ◽

M. A. Zainal Abidin ◽

M. Y. Wong ◽

F. Abed Ashtiani

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Leaf Tissue ◽

Gray Leaf Spot ◽

Universal Primers ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Morphological Criteria ◽

Pepper Leaves

Symptoms of gray leaf spot were first observed in June 2011 on pepper (Capsicum annuum) plants cultivated in the Cameron Highlands and Johor State, the two main regions of pepper production in Malaysia (about 1,000 ha). Disease incidence exceeded 70% in severely infected fields and greenhouses. Symptoms initially appeared as tiny (average 1.3 mm in diameter), round, orange-brown spots on the leaves, with the center of each spot turning gray to white as the disease developed, and the margin of each spot remaining dark brown. A fungus was isolated consistently from the lesions using sections of symptomatic leaf tissue surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto PDA and V8 agar media (3). After 7 days, the fungal colonies were gray, dematiaceous conidia had formed at the end of long conidiophores (19.2 to 33.6 × 12.0 to 21.6 μm), and the conidia typically had two to six transverse and one to four longitudinal septa. Fifteen isolates were identified as Stemphylium solani on the basis of morphological criteria described by Kim et al. (3). The universal primers ITS5 and ITS4 were used to amplify the internal transcribed spacer region (ITS1, 5.8, and ITS2) of ribosomal DNA (rDNA) of a representative isolate (2). A 570 bp fragment was amplified, purified, sequenced, and identified as S. solani using a BLAST search with 100% identity to the published ITS sequence of an S. solani isolate in GenBank (1). The sequence was deposited in GenBank (Accession No. JQ736024). Pathogenicity of the fungal isolate was tested by inoculating healthy pepper leaves of cv. 152177-A. A 20-μl drop of conidial suspension (105 spores/ml) was used to inoculate each of four detached, 45-day-old pepper leaves placed on moist filter papers in petri dishes (4). Four control leaves were inoculated similarly with sterilized, distilled water. The leaves were incubated at 25°C at 95% relative humidity for 7 days. Gray leaf spot symptoms similar to those observed on the original pepper plants began to develop on leaves inoculated with the fungus after 3 days, and S. solani was consistently reisolated from the leaves. Control leaves did not develop symptoms and the fungus was not reisolated from these leaves. Pathogenicity testing was repeated with the same results. To our knowledge, this is the first report of S. solani causing gray leaf spot on pepper in Malaysia. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. P. S. Camara et al. Mycologia 94:660, 2002. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) B. M. Pryor and T. J. Michailides. Phytopathology 92:406, 2002.

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First Report of Black Spot of Acanthus ilicifolius Caused by Fusarium solani in China

Plant Disease ◽

10.1094/pdis-04-14-0432-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1438-1438 ◽

Cited By ~ 1

Author(s):

H.-R. Su ◽

H. He ◽

Q.-Z. Huang ◽

N.-H. Lu ◽

Y.-B. Zhang

Keyword(s):

Fusarium Solani ◽

Morphological Characteristics ◽

Black Spot ◽

Morphological Observation ◽

Molecular Characteristics ◽

Beta Tubulin ◽

First Report ◽

Acanthus Ilicifolius ◽

Black Spots ◽

Initial Symptoms

Acanthus ilicifolius (family Acanthaceae) grows mainly in tropical coastal areas and is an important medicinal plant that can be used to treat asthma, rheumatism, etc. In July 2013, symptoms of black spots on the leaves of A. ilicifolius were observed in the Mangrove Conservation Area of Shenzhen Futian (22°32′ N, 114°03′ E) and Leizhou peninsula (20°12′~21°35′ N, 109°30′~110°55′ E), Guangdong Province, China. Initial symptoms of the disease were a small, dark brown spots (4 to 5 × 4 to 6 mm) surrounded by a yellow halo (1 to 2 mm in diameter), that would later extend to round or irregular black spots. Leaves eventually turned chlorotic and plants defoliated. Tissues from symptomatic leaves were excised, surface sterilized with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% HgCl2 solution for 45 s, rinsed three times in sterile water, cut into small pieces (2 to 3 mm), plated on potato dextrose agar (PDA), and incubated 3 to 5 days at 28°C without light. Four isolates named from LSL-1 to LSL-4 with different morphological characteristics were obtained. To fulfill Koch's postulates, wounded and non-wounded leaves were inoculated. Fresh wounds were made with a sterile needle on detached leaves and on living plants. Mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. For the control, the healthy leaves were inoculated with PDA plugs. All treatments were incubated at room temperature. Black spots were observed on the wounded leaves inoculated with isolate LSL-1 after 3 days, while the other three isolates and the control remained symptomless, and the pathogen similar to LSL-1 was re-isolated from the diseased leaves. Non-wounded leaves didn't become infected. The pathogenic test was repeated three times with the same conditions, and it was confirmed that LSL-1 was the pathogen causing the black spot of A. ilicifolius. Identification of the pathogen was conducted using morphological and molecular characteristics. Hyphal tips of LSL-1 were transferred to PDA medium in petri dishes for morphological observation. Two types of conidia were observed. The macroconidia were cylindrical to slightly curved, falciform shaped, with two to four septa, and measured 39 to 45 × 4.7 to 5.0 μm. The microconidia were oval to kidney shaped, single celled, 8 to 10 × 2.5 to 3.5 μm. Chlamydospores were also observed, produced singly or in pairs. Based on morphology (1,4), the isolate was tentatively identified as Fusarium solani. For molecular identification, the internal transcribed spacer (ITS) of ribosomal DNA, beta-tubulin gene, and translation elongation factor 1-alpha (EF-1α) gene was amplified using the ITS1/ITS4 (5), ITS4/ITS5 (5), T1/T2 (2) and EF1/EF2 (3) primer pairs. The gene sequences were deposited in GenBank (KJ720639 for the ITS1/ITS4 region, KF826493 for the ITS4/ITS5 region, KJ720638 for the beta-tubulin, and KF826492 for EF-1α region) and showed 99% identity to the F. solani strains (AY633746 for ITS1/ITS4 region, AM412637 for ITS4/ITS5 region, KF255996 for beta-tubulin region, DQ246859 for EF-1α region). According to these results, the pathogen of black spot of A. ilicifolius was identified as F. solani. To the best of our knowledge, this is the first report of F. solani causing black spot of A. ilicifolius in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) B. A. Pérez et al. Plant Dis. 91:1053, 2007. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

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First Report of Neofusicoccum parvum Causing Dieback Disease of Chinese Weeping Cypress in China

Plant Disease ◽

10.1094/pdis-94-5-0641c ◽

2010 ◽

Vol 94 (5) ◽

pp. 641-641 ◽

Cited By ~ 3

Author(s):

S. B. Li ◽

J. Z. Li ◽

S. C. Li ◽

Z. H. Lu ◽

J. H. Wang ◽

...

Keyword(s):

Cross Sections ◽

Elongation Factor ◽

Aerial Mycelium ◽

Central China ◽

Internal Transcribed Spacer Region ◽

Middle Point ◽

Neofusicoccum Parvum ◽

First Report ◽

Dieback Disease ◽

And Control

Cupressus funebris Endl. (Chinese weeping cypress) is native to southwestern and central China. In June 2008, blighted shoots of Chinese weeping cypress trees were observed in Yunnan Province (southwestern China). Symptomatic trees were located in an ornamental planting established approximately 8 to 12 years ago. Additional samples were collected from 11 locations in the provinces of Sichuan, Yunnan, Guizhou, and Chongqing. Disease symptoms included yellowing and wilting of leaves on several branches, followed by sudden death within 6 to 8 weeks. Cross sections on trunks and branches revealed darkened zones. Tissue from diseased samples was plated on potato dextrose agar (PDA) and incubated at 25°C. Fungal isolates developed copious, white, aerial mycelium that became dark gray after 4 to 6 days and formed black pycnidia after 25 days. Conidia were hyaline, ellipsoidal to fusiform, externally smooth, thin walled, nonseptate, and measured 12.5 to 18.5 × 4.0 to 6.5 μm. Identity was confirmed by analysis of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2) and the translation elongation factor 1-alpha (EF1-α). BLAST searches at GenBank showed a high identity with reference sequences (ITS: >99%; EF1-α: 100%). Representative sequences of both regions were deposited in GenBank (ITS: Accession No. FJ842960 and FJ842961; EF1-α: Accession No. GU811148). Morphological and molecular results confirmed this species as Neofusicoccum parvum, reported as the anamorph of Botryosphaeria parva. Pathogenicity tests were conducted by stem inoculation of 2-year-old C. funebris seedlings. Mycelial plugs (4 mm in diameter) of N. parvum from actively growing colonies were applied to same-size bark wounds on the middle point of the stems. Control seedlings were inoculated with sterile PDA plugs. Inoculated and control seedlings (five each) were kept in a greenhouse and watered as needed. After 5 weeks, all C. funebris seedlings showed leaf wilting and dark vascular stem tissue. N. parvum was reisolated from all inoculated, symptomatic tissues, fulfilling Koch's postulates; no symptoms were visible in the control seedlings. N. parvum has previously been reported to cause canker and dieback disease of avocado (3), mango (2), and magenta cherry (Syzygium paniculatum) (1). To our knowledge, this is the first report of N. parvum causing dieback of C. funebris in China. References: (1) R. C. Ploetz et al. Plant Pathol. 58:801, 2009. (2) B. Slippers et al. Mycologia 97:99, 2005. (3) T. Zea-Bonilla et al. Plant Dis. 91:1052, 2007.

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First Report of the Anamorph of Glomerella acutata Causing Anthracnose on Avocado Fruits in Mexico

Plant Disease ◽

10.1094/pdis-91-9-1200a ◽

2007 ◽

Vol 91 (9) ◽

pp. 1200-1200 ◽

Cited By ~ 4

Author(s):

G. Avila-Quezada ◽

H. V. Silva-Rojas ◽

D. Teliz-Ortiz

Keyword(s):

Colletotrichum Gloeosporioides ◽

Soft Rot ◽

Lower Surface ◽

Persea Americana ◽

Colletotrichum Acutatum ◽

Morphological Identification ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Mexican State ◽

Pathogenicity Tests

Mexico is a major avocado (Persea americana) producer in the world. Glomerella cingulata (anamorph Colletotrichum gloeosporioides) has been reported as a causal agent of anthracnose on avocado fruits worldwide (3), while G. acutata (anamorph Colletotrichum acutatum) has been identified as the cause of this disease only in New Zealand (2) and Australia (4). This study was done with the objective to determine the Glomerella spp. involved as the causal agents of avocado anthracnose in Mexico. From 2003 to 2006, avocado fruits cv. Hass with anthracnose symptoms appearing as brown-black lesions on the pericarp and soft rot in the mesocarp were collected in 10 counties in Michoacan, the leading avocado-producing Mexican state. Glomerella spp. were isolated on potato dextrose agar (PDA) for molecular and morphological identification. A phylogenetic analysis was done by amplifying the internal transcribed spacer region of rDNA for 28 of the isolates. Primers ITS5/NL4 was used and successfully amplified bands of approximately 1,000 bp. Each sequence corresponding to Glomerella spp. was compared with sequences deposited in the GenBank database using BLAST. The results from molecular approach indicated 86% of the isolates used in this study were G. cingulata and 14% were G. acutata. Sequences of both species were deposited in GenBank under Accession Nos. EF221828, EF221829, and EF221830 for G. cingulata and EF175780, EF221831, and EF221832 for G. acutata. Colonies of G. acutata that developed on PDA medium were pale gray, occasionally the lower surface was olive green, and the center was covered with orange-to-salmon pink masses of conidia and perithecia. Conidia grown in the same media were straight, fusiform, 8.2 to 16.5 μm long, and 2.7 to 4.0 μm wide (4). Pathogenicity tests of G. acutata were carried out by inoculating six healthy cv. Hass fruits (1) at three evenly spaced locations on the fruit surface with a needle dipped in a conidial mass from a 3-day-old monoconidial culture of G. acutata. Fruits were then incubated in a moist chamber for 3 days. Anthracnose symptoms were observed on healthy fruits inoculated with G. acutata, while control fruits inoculated with sterile water did not develop symptoms. The fungi were reisolated successfully to confirm the pathogen's identity using morphological key. To our knowledge, this is the first report of G. acutata causing anthracnose on avocado fruits in Mexico. References: (1) R. Guetsky et al. Phytopathology 95:1341, 2005. (2) W. F. T. Hartill. N. Z. J. Crop Hortic. Sci. 19:297, 1991. (3) D. Prusky. Annu. Rev. Phytopathol. 34:413, 1996. (4) J. H. Simmonds. Qld. J. Agric. Anim. Sci. 22:437, 1965.

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