Black Leaf Spot Research Articles

Fungi associated with leaves of Sonneratia apetala Buch. Ham and Sonneratia caseolaris (L.) Engler from Rangabali coastal zone of Bangladesh

A total of six species and one genus of fungi associated with black leaf spot of Sonneratia apetala Buch. Ham (Kewra); and anthracnose and small leaf spot of S. caseolaris (L.) Engler (Choila) were isolated following “Tissue planting” method. The fungi associated with black spot of S. apetala were Aspergillus fumigatus Fresenius, Colletotrichum lindemuthianum (Sacc. & Magn.) Br. & Cav., Pestalotiopsis guepinii (Desm.) Stey. and Phoma betae Frank. Anthracnose of S. caseolaris showed the association of A. fumigatus and P. guepinii. The fungi associated with leaf spot of S. caseolaris were Curvularia fallax Boedijn, Fusarium Link, Penicillium digitatum (Pers.) Sacc. and P. betae. Frequency percentage of association of P. guepinii was highest (74.10) in black spot of S. apetala whereas the same fungus showed highest frequency percentage (85.70) in case of anthracnose of S. caseolaris. Phoma betae showed highest frequency percentage (60.00) in leaf spot of S. caseolaris. Phoma betae is first time recorded from Bangladesh.
 Dhaka Univ. J. Biol. Sci. 27(2): 155-162, 2018 (July)

Identification of Fusarium proliferatum causing leaf spots on Cymbidium orchids in Taiwan

AbstractPrevious research indicated that black and yellow leaf spots on Cymbidium, Ondontioda, Dendrobium and Cattleya could be caused by Fusarium proliferatum worldwide. However, the agent causing leaf spot on Cymbidium spp. plants is still obscure in Taiwan. Thirty‐five F. fujikuroi species complex (FFSC)‐like isolates were collected from Cymbidium leaf spot from different greenhouses in Taiwan. All isolates were identified as F. proliferatum based on morphological characteristics and molecular analysis. Sequence of translation elongation factor 1‐alpha gene showed 99%–100% homology with F. proliferatum. In addition, two assay techniques using either detached leaves or seedlings were used to evaluate the pathogenicity and host range of the isolates and consequently their effects on Cymbidium and other orchid plants. Pathogenicity assays revealed that all isolates induced black and necrotic spots on detached leaves of Cymbidium, showing 9.4%–29.5% severity on seedlings of Cymbidium. Results of host specificity tests on detached leaves of different plants indicated that the F. proliferatum isolates collected from Cymbidium plants caused severe black spots on Oncidium, Cymbidium, Dendrobium and Cattleya plants. The symptoms on Phalaenopsis plants were relatively mild. Results of host specificity tests on plant seedlings indicated that the F. proliferatum isolates of Cymbidium origin were also pathogenic to Oncidium, Cymbidium and Dendrobium, but not to Cattleya and Phalaenopsis. Phylogenetic analysis of the translation elongation factor (TEF) gene among all fungal isolates using maximum parsimony (MP) and Bayesian inference (BI) methods revealed that the isolates of F. proliferatum from Cymbidium spp. could be separated from other FFSC‐like species with high phylogenetic support.

Morphological and molecular characterization of the strawberry black leaf spot pathogen referred to as the strawberry pathotype of Alternaria alternata

Abstract The remaining unclarified taxon among the seven known pathotypes of host-selective toxin (HST)-producing Alternaria alternata, namely, the strawberry pathotype (the strawberry black leaf spot pathogen), is taxonomically revised and re-described herein. According to our morphological observations, reference isolates of strawberry and Japanese pear pathotypes, which are toxic to leaves of Japanese pear ‘Nijisseiki’, have conidia that are formed in chains of 3–13, usually without lateral branches, after 7 d incubation on potato-carrot agar. The mean size of the conidia is 27–31 × 11–13 μm. Morphological characteristics of the examined isolates are identical to those of A. gaisen rather than A. alternata. A phylogenetic tree obtained by analysis of a combined dataset of ITS, gapdh, rpb2, tef1, Alt a 1, and endoPG sequences also strongly supports both pathotypes as one species, A. gaisen. We re-describe the fungus as A. gaisen Nagano ex Bokura and propose two formae speciales of the species, A. gaisen f. sp. fragariae producing AF-toxin and f. sp. pyri producing AK-toxin. The epitype specimen and ex-epitype culture of A. gaisen are newly designated.

First report of Epicoccum nigrum causing disease in Lotus corniculatus in Argentina

Lotus corniculatus is a species adapted to field conditions in the most important cattle production region in Argentina and constitutes a very valuable forage species. In January 2018, black leaf spots were observed on L. corniculatus (Fig. 1) in field plots in Chascomús, Buenos Aires. These spots were amphigenous, circular to irregular, 2-8 mm diameter, close to the leaf margin and reddish brown. Single lesions often coalesced to form larger lesions and became dark brown (Fig. 1). Fungal isolates were obtained from the shoot lesions and were incubated on oatmeal agar, malt extract and potato dextrose agar (Crous et al., 2) at 25°C in darkness. Macroscopic features of colonies on these media and mycelia characteristic were characterised under a microscope (Fig. 2) and conformed morphologically most closely to the genus Epicoccum (Chen et al., 1). Total genomic DNA was extracted from fresh mycelia and PCR amplification was done using the ITS4 and ITS5 primers (White et al., 5) designed to amplify the internal transcribed spacer (ITS) region. The amplified PCR products were purified and sequenced (GenBank Accession No. MH042300). A BLAST search revealed 99% identity to E. nigrum (KX815296 and MF510615). An inoculation trial was done as follows. Lotus corniculatus plants were grown in a growth chamber at 25°C at 30-40% relative humidity with a 12 hr photoperiod. Thirty-day-old leaves were placed on Petri plates containing water agar medium. Leaves were sprayed with a conidia solution prepared by agitating the fungal lawn with distilled water in a petri dish culture, then suspending in 0.05% Tween 20 to a final concentration of 105 conidia/ml. Control leaves were sprayed with water containing 0.05% Tween 20. Petri plates were maintained at 25°C, 100% relative humidity for 16 days. The inoculated L. corniculatus plants were observed to be wilted with a yellow appearance and black spots (Fig. 3). The causal fungus was isolated and the macro- and microscopic characteristics were consistent with the inoculated fungus. The genus Epicoccum was reported in Argentina as a seed pathogen in Lotus spp. (Sisterna & Lori, 4). The isolate in the current study had 98% identity with E. nigrum (KC568289 and KY303832) that caused leaf spot in Lablab purpureus and brown leaf spot in loquat, respectively (Mahadevakumar et al., 3; Wu et al., 6). To our knowledge, this is the first report of E. nigrum causing leaf spot in L. corniculatus in Argentina.

Transcriptomics Analysis of the Chinese Pear Pathotype of Alternaria alternata Gives Insights into Novel Mechanisms of HSAF Antifungal Activities.

Alternaria alternata (Fries) Keissler is a lethal pear pathogen that causes leaf black spot disease of pear in Southern China. Heat-stable activity factor (HSAF) is a polycyclic tetramate macrolactam (PTM) produced by Lysobacter enzymogenes and many other microbes with a broad-spectrum antifungal activity against many filamentous fungi. In this study, we evaluated the antifungal effect of HSAF against A. alternata and proposed its antifungal mechanism in A. alternata. We report that HSAF inhibited the mycelial growth of A. alternata in a dose-dependent manner. Transcriptomics analysis revealed that HSAF treatment resulted in an expression alteration of a wide range of genes, with 3729 genes being up-regulated, and 3640 genes being down-regulated. Furthermore, we observed that HSAF treatment disrupted multiple signaling networks and essential cellular metabolisms in A. alternata, including the AMPK signaling pathway, sphingolipid metabolism and signaling pathway, carbon metabolism and the TCA (tricarboxylic acid) cycle, cell cycle, nitrogen metabolism, cell wall synthesis and a key hub protein phosphatase 2A (PP2A). These observations suggest that HSAF breaches metabolism networks and ultimately induces increased thickness of the cell wall and apoptosis in A. alternata. The improved understanding of the antifungal mechanism of HSAF against filamentous fungi will aid in the future identification of the direct interaction target of HSAF and development of HSAF as a novel bio-fungicide.

Validation of Inoculation test and Screening for Resistance Sources of Mustard-Rapeseed Cultivars in Bangladesh Against Alternaria brassicicola

Alternaria leaf spot or black leaf spot caused by Alternaria brassicicola is the devastating diseases of mustard-rapeseed in Bangladesh, and can cause yield loss up to 60% every year. A total of 27 cultivars of Brassica spp. where Brassica rapa (13), B. juncea (5) and B. napus (9) were selected for resistance screening against A. brassicicola. Two inoculation methods viz. detached leaf and seedling inoculation were assessed for checking the validation of inoculation technique using cultivated mustard-rapeseed varieties in Bangladesh a method to measure resistance to A. brassicicola. A significant positive correlation between the results of two inoculation methods was found in this study. The detached leaf technique was more suitable due to development of clear symptoms on the leaves within 36 hrs, and suitable for screening large scale genotypes for resistance. The 3rd leaves of 30 day-old were more suitable for inoculation having severe symptoms than the 4th leaves. Among 27 Bangladeshi mustard-rapeseed cultivars all cultivars expressed susceptible reaction to A. brassicicola, except BINA Sharisha-8, as it had border line resistance.

Vegetative compatibility grouping of Alternaria brassicicola causing black leaf spot in cauliflower

A study was conducted to understand the relationship between pathogenicity and vegetative compatibility groups (VCG) of thirty-two A. brassicicola isolates causing black leaf spot in cauliflower. Nine isolates were highly pathogenic as the spot size produced by them are > 1 cm in diameter where as eight isolates were moderately pathogenic as they produced spot size of 0.6–1 cm and fifteen isolates were least pathogenic as the spot size were 0.2–0.5 cm in diameter. One hundred forty-four chlorate-resistant mutants were isolated from A. brassicicola isolates on water agar and PDA both contains 3% potassium chlorate whose number varied from 1 to 7. These mutants were further classified into three different phenotypes: nit M, nit 1, and nit 3. The largest group of mutants proved to nit 1 (36.8%), followed by nit 3 (32.6%), nit M (15.3%), wild type (9.3%) and unknown (6.0%). The anastomosis between nit mutants were also observed under microscope. Based on complementary reactions these thirty-two isolates were grouped into five arbitrary designated VCG which can be further used for the diversity analysis of A. brassicicola isolates.

Co-inoculation of lucerne (Medicago sativa) with an AM fungus and a rhizobium reduces occurrence of spring black stem and leaf spot caused by Phoma medicaginis

Spring black stem and leaf spot of lucerne (alfalfa, Medicago sativa L.), caused by Phoma medicaginis, is an important disease in temperate regions of the world. It is now a serious disease threatening global lucerne production. This experiment was designed to test the combined effects of the arbuscular mycorrhizal (AM) fungus Funneliformis mosseae and the rhizobium Sinorhizobium medicae on growth, nutrient uptake and disease severity in lucerne. The results showed that F. mosseae increased plant phosphorus and nitrogen uptake and plant dry weight, and this beneficial effect was enhanced when in association with S. medicae. Rhizobial and AM fungal effects were mutually promoting; inoculation with AM fungus significantly increased the formation of root nodules, and inoculation with rhizobium increased the percentage of root length colonised by AM fungus (P < 0.05). After infection with P. medicaginis, typical leaf spot symptoms with the lowest disease incidence and disease index occurred on plants that were host to both F. mosseae and S. medicae. Plants with both symbiotic microorganisms had higher activities (concentrations) of phenylalanine ammonia-lyase, chitinase, β-1,3-glucanase, lignin, hydroxyproline-rich glycoprotein and jasmonic acid. Therefore, the tested AM fungus (F. mosseae) and rhizobium (S. medicae) have the potential to reduce damage and yield loss in lucerne from spring black stem and leaf spot caused by P. medicaginis.

A noncanonical poly(A) RNA polymerase gene affects morphology in Phoma medicaginis

Phoma medicaginis (syn. Ascochyta medicaginicola Qchen & L. Cai) causes spring black stem and leaf spot, an important disease of alfalfa and annual medics. P. medicaginis forms uninucleate conidia in melanized pycnidia and is genetically tractable using Agrobacterium mediated transformation (ATMT), resulting in random integration of T-DNA that occasionally generates pycnidial mutants. The T-DNA tagged mutant, P265 displayed smaller pycnidia and more aerial hyphae than the wild type. A single T-DNA disrupted a putative noncanonical poly(A) RNA polymerase gene, Pmncpap1, which in yeast interacts with ribonucleotide reductase (RNR). As in yeast mutants, P265 showed sensitivity to hydroxyurea (HU), a RNR inhibitor. To characterize the role of Pmncpap1, targeted ΔPmncpap1 mutants were created using a hygromycin selectable marker flanked by 1 Kbp regions of Pmncpap1. ΔPmncpap1 mutants possessed similar morphological features to those of P265. The plasmid for rescue of PmncPAP1, pCAM−Nat1 (nourseothricin selection) was constructed and used to introduce full-length PmncPAP1 into mutants. Rescued P265 showed partial recovery of wild type and the original T-DNA was lost due to homologous integration. To our knowledge, this is the first ncPAP to be examined in a filamentous fungus.

First Report of Strawberry Leaf Spot Caused by Alternaria alternata in Pakistan

Strawberry (Fragaria × ananassa Duch.) has recently become an economically important fruit crop in Pakistan and is cultivated on approximately 1500 ha. A survey across Punjab, Khyber Pakhtunkhwa (KPK) provinces and Islamabad (Islamabad Capital territory) was conducted during March and April of 2015-2016 growing season revealed black leaf spot symptoms on strawberry leaves. Leaf spots were irregular in shape, 3 to 6 mm in diameter, and blackish brown. Disease incidence was found 17 to 55 % in visited fields. Portions of symptomatic leaf tissue (3 × 5 mm) were excised from the margins of lesions; surface disinfected with 2% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water (SDW) and blotted dry. Leaf tissue was then placed on Czapek Dox agar (CDA) and incubated at 25 ± 2°C with a 12-h photoperiod for 3 to 6 days. Morphology of the 78 isolates was examined; colonies were initially white, then turned light to dark brown and greyish black and produced abundant aerial hyphae. Conidiop...

Incidence Rates of Major Diseases of Kiwiberry in 2015 and 2016.

Incidence rates of diseases in kiwiberry orchards were investigated monthly from late June to late September in Gwangyang and Boseong in 2015 and 2016. The impact of postharvest fruit rot was investigated during ripening after harvest. Bacterial canker was only observed on one single tree in 2015, but black rot, powdery mildew, leaf spot and blight, and postharvest fruit rot diseases were problematic throughout the study period in both 2015 and 2016. Incidence rates of the diseases varied with kiwiberry cultivar, region and sampling time. Incidence rates of powdery mildew, leaf spot and blight diseases increased significantly during the late growing stages near fruit harvest, while black rot peaked in late August. Incidence rate of postharvest fruit rot on fruit without fruit stalks was less than half of fruit with fruit stalks, regardless of kiwiberry cultivars. Among the four cultivars, Mansu was relatively resistant to black rot and postharvest fruit rot diseases. In our knowledge, this is the first report of various potential pathogens of kiwiberry in Korea.

Biocontrol Potential of Streptomyces hydrogenans Strain DH16 toward Alternaria brassicicola to Control Damping Off and Black Leaf Spot of Raphanus sativus.

Biocontrol agents and their bioactive metabolites provide one of the best alternatives to decrease the use of chemical pesticides. In light of this, the present investigation reports the biocontrol potential of Streptomyces hydrogenans DH16 and its metabolites towards Alternaria brassicicola, causal agent of black leaf spot and damping off of seedlings of crucifers. In vitro antibiosis of strain against pathogen revealed complete suppression of mycelial growth of pathogen, grown in potato dextrose broth supplemented with culture supernatant (20% v/v) of S. hydrogenans DH16. Microscopic examination of the fungal growth showed severe morphological abnormalities in the mycelium caused by antifungal metabolites. In vivo studies showed the efficacy of streptomycete cells and culture supernatant as seed dressings to control damping off of Raphanus sativus seedlings. Treatment of pathogen infested seeds with culture supernatant (10%) and streptomycete cells significantly improved seed germination (75–80%) and vigor index (1167–1538). Furthermore, potential of cells and culture supernatant as foliar treatment to control black leaf spot was also evaluated. Clearly visible symptoms of disease were observed in the control plants with 66.81% disease incidence and retarded growth of root system. However, disease incidence reduced to 6.78 and 1.47% in plants treated with antagonist and its metabolites, respectively. Additionally, treatment of seeds and plants with streptomycete stimulated various growth traits of plants over uninoculated control plants in the absence of pathogen challenge. These results indicate that S. hydrogenans and its culture metabolites can be developed as biofungicides as seed dressings to control seed borne pathogens, and as sprays to control black leaf spot of crucifers.

Occurrence of Cabbage Black Leaf Spot in El-Sharkia Governorate during 2010-2011 Growing Season

Occurrence of Cabbage Black Leaf Spot in El-Sharkia Governorate during 2010-2011 Growing Season

Biological Control of Alternaria brassicicola Causing Cabbage Black Leaf Spot Disease

Alternaria black leaf spot disease appeared to be wide spread incabbage fields of El-Sharkia governorate, Egypt during 2010-2011 growing season. Alternaria brassicicola was the most frequentlyisolated fungus. Different bioagent microorganisms, isolated from thephyllosphere of the resistant cabbage cultivar (OS-Cross) wereinvestigated microscopically for their antagonistic effect against thepathogen in vitro. Trichoderma spp., Bacillus subtilis and B1 bacterialisolates as well as their culture filtrates proved to be highly effectivein inhibiting A. brassicicola spore germination as well as causingmycelium and spore malformation subsequently reducing the diseaseincidence and severity when applied in vivo.

Characterization of Colletotrichum fructicola, a new causal agent of leaf black spot disease of sandy pear (Pyrus pyrifolia)

In recent years, a devastating fungal disease characterized by small black spots (<1 mm) on sandy pear (Pyrus pyrifolia Nakai) leaves has occurred in sudden outbreaks in southern China, resulting in severe defoliation and a loss of fruit quality and yield. To identify the etiology of the disease, 147 fungal isolates from sandy pear leaves showing typical black spots were collected from 30 orchards; 10 fungal species were included among the isolates, and the prevailing 124 Colletotrichum isolates were subjected to morphological and molecular characterization. Based on differences in colony morphology, the isolates were separated into five groups (I to V); the groups were specifically separated according to a combination of colony color, pigment, sporulation, perfect stage, and spore size. In addition to their morphological features, the five groups were assessed for their molecular and taxon statuses based on ITS sequences, multilocus regions (ITS, ACT, TUB, CHS-1, GAPDH), and the Apn2/MAT locus, which provided molecular proof for the identification of these isolates as Colletotrichum fructicola Prihastuti. The result also indicated different resolution of the three loci in delimiting the species. Koch’s postulates were fulfilled by inoculating conidium suspensions of the representative isolates on attached and detached leaves of sandy pear cvs. Cuiguan, Xueqing and Huali No. 2 with analogous conditions (under saturated humidity and with tiny wounds) to those that appeared in the field, and similar symptoms were elicited by the isolates, suggesting that C. fructicola was causing the disease. To our knowledge, this is the first report of C. fructicola causing leaf black spot of pear. The etiological clarification and characterization of the C. fructicola isolates provide useful information to aid in the understanding of leaf black spot disease in pears and for designing management strategies to control this economically significant disease.

First Report of Black Leaf Spot Caused byAlternaria alternataon Ramie in China

AbstractIn 2012 and 2013, black leaf spot disease was observed on ramie plants in Hunan and Hubei Provinces, China. In the field, the symptoms of this disease included dark green to black big spots on leaves, often resulting in upwardly curled leaf margins. The pathogen isolates were identified asAlternaria alternata(Fr.) Keissler on the basis of morphology and sequence similarity of 99–100% to the published data for internal transcribed spacer (ITS), and glyceraldehyde‐3‐phosphate dehydrogenase (gdp). To our knowledge, this is the first report ofAlternarialeaf spot of ramie in China.

Suppression of rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases by Meyerozyma guilliermondii TA-2 and the nature of protection

This study was conducted to evaluate the efficacy of yeast strain TA-2 for controlling rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases. Microscopic and phylogenetic analyses based on rDNA-internal transcribed region (ITS) and rDNA-D1/D2 sequences indicated that yeast strain TA-2 is Meyerozyma guilliermondii. Pretreatment with TA-2 by soil drenching significantly reduced the severity of black leaf spot disease caused by Alternaria brassicicola and leaf blast disease caused by Magnaporthe oryzae. Symptom development of tomato bacterial wilt caused by Ralstonia solanacearum in both soil drench and needle inoculation tests was significantly reduced in TA-2-pretreated plants under soil drenching. Disease severity and R. solanacearum growth were significantly reduced in tomato plants pretreated with yeast culture, cell suspension, or culture filtrate of TA-2 under soil drenching. TA-2 does not produce antibiotics. The present study indicates that disease suppression is systemic, as the roots were treated with TA-2 and the pathogens were inoculated onto leaves or stems, thereby separating the two spatially. M. guilliermondii TA-2 could become a promising natural antimicrobial agent against rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases and might be useful as an eco-friendly control measure, contributing to sustainable agriculture.

First Report of Alternaria alternata Causing Black Leaf Spot of Rubber Tree in China.

We first reported Alternaria heveae (E.G. Simmons ) to be the pathogen that caused black leaf spot of rubber tree (Hevea brasiliensis Muell. Arg) in Heikou county in July 2014 (1). Black leaf spots that resembled the symptoms caused by A. heveae were observed on the leaves of rubber trees of the whole propagule collection nursery in Jingping County (22°68' N and 103°05' E) of Yunnan Province. Black foliar spots (0.1 to 2 mm in diameter) surrounded by a yellow halo with lesions slightly sunken on the leaf surface were observed. To confirm whether the disease was caused by the same pathogen, 5-mm2 sections were removed from the leading edge of the lesion and were surface-sterilized in 75% ethanol, air-dried, plated on potato carrot agar (PCA), and incubated at 28°C in the dark. Colonies of the fungus on PCA had round margins and little aerial mycelia with gray-black coloration after 6 days of growth on PCA (2). Medium brown conidia were found to be in short chains of two to eight spores, ovoid, obclavate, and obpyriform, with or without a short conical or cylindrical-shaped apical beak. Conidia ranged from 22.5 to 67.5 μm long (mean 39.9 μm) × 10 to 15 μm wide (mean 12.5 μm; 100 colodia were measured), with three to six transverse septa and zero to three longitudinal or oblique septa. Morphological characteristics matched the descriptions of A. alternata [(Fries) Keissler] (4).The ITS1-5.8S-ITS2 region of one single-spore isolate, Ah02JP1, was amplified with primers ITS1 and ITS4. The PCR product was sequenced directly and deposited in GenBank (Accession No. KM111289). A BLAST search of the GenBank database revealed 100% similarity with A. alternata isolates KJ829535.1, KJ677246.1, and KF813070.1. Therefore, the pathogen was identified as A. alternata on the basis of its morphological characteristics and ITS sequence. Pathogenicity of a representative isolate, Ah02JP1 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage. Leaves were spray-inoculated (104 conidia per milliliter spore suspension) until drops were equally distributed using a manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags to maintain high relative humidity. The plastic bags were removed 2 days post-inoculation (dpi), and the plants were monitored daily for symptom development. Five days post-inoculation, spots similar to the original ones seen on the field trees developed on all inoculated leaves, while control leaves remained symptomless. A. alternata was re-isolated from spray-inoculated leaves, confirming Koch's postulates. A. alternata has been reported as the causal agent of leaf blight of rubber tree in India, which initially appeared as minute spots on leaves and enlarged with the growth of the leaves (3). However, in the present study, the symptoms (black leaf spots) remained small over time after inoculation. To our knowledge, this is the first report of A. alternata on rubber tree in China. Correct identification of pathogens is essential for disease management strategies. This report will establish a foundation for the further study of Alternaria alternata to address the disease effectively.

FIRST REPORT OF FUSARIUM BRACHYGIBBOSUM CAUSING LEAF SPOT ON OLEANDER IN IRAN

Oleander (Nerium oleander) is an evergreen ornamental extensively grown in Iran. In April 2013, dark brown to black leaf spots were observed on oleander leaves in the Yazd province. As disease progressed, leaf spots increased from 10 to 20 mm in diameter and changed from circular to elliptical in shape. Three leaf spots were excised, surface-sterilized in 0.5% sodium hypochlorite and placed on potato dextrose agar (PDA). Colonies were initially white, then turned from orange-yellow to medium red with abundant aerial mycelium. Macroconidia were rare and scattered (mean of 20 spores: 25.92×4.43 μm) with three to four septa; microconidia were slightly curved, ovoid, and fusiform (mean of 20 spores: 11.05×3.97 μm) with zero to two septa. Spherical chlamydospores (mean of 20 spores: 11.01 μm) were terminal and intercalary, single or in chains. Based on these characteristics, the fungus was identified as Fusarium brachygibbosum Padwick (Al-Sadi et al., 2012). Pathogenicity of the fungal isolate was proven by placing seven-day-old mycelial plugs (5 mm) grown on PDA on oleander leaves. Eight days after inoculation, leaf spots developed on the inoculated plants from which F. brachygibbosum was re-isolated. Identification of the fungus was confirmed by comparing the sequences generated from the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS4 and ITS5 primers) (White et al., 1990). These sequences shared 100% nucleotide similarity with those of F. brachygibbosum (GenBank accession No. KF985966). This pathogen has previously been reported on date palm (Padwick, 1945) and as the causal agent of dieback on Euphorbia larica (Al-Mahmooli et al., 2013). To our knowledge, this is the first report of F. brachygibbosum on oleander.

First Report of Alternaria heveae Causing Black Leaf Spot of Rubber Tree in China.

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop of tropical areas for natural rubber production. In October 2013, foliar spots (0.1 to 0.4 mm in diameter), black surrounded by a yellow halo, and with lesions slightly sunken were observed on the rubber tree leaf in a growing area in Heikou County of Yunnan Province. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on PDA plates, and incubated at 28°C with alternating day/night cycles of light. The pathogen was observed growing out of many of the leaf pieces, and produced abundant conidia. Colonies 6.1 cm in diameter developed on potato carrot agar (PCA) after 7 days, with well-defined concentric rings of growth. Colonies on PCA were composed of fine, dark, radiating, surface and subsurface hyphae. Conidia produced in PCA culture were mostly solitary or in short chains of 2 to 5 spores, long ovoid to clavate, and light brown, 40 to 81.25 × 8 to 20 μm (200 colonies were measured), with 3 to 6 transverse septa and 0 to 2 longitudinal or oblique septa. Morphological characteristics were similar to those described for Alternaria heveae (3,4). A disease of rubber tree caused by Alternaria sp. had been reported in Mexico in 1947 (2). DNA of Ah01HK13 isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers was completed. From the BLAST analysis, the sequence of Ah01HK13 (GenBank Accession No. KF953884), had 97% similarity to A. dauci, 96% identical to A. macrospora (AY154701.1 and DQ156342.1, respectively), indicating the pathogen belonged to Alternaria genus. According to morphological characteristics, this pathogen was identified as A. heveae. Pathogenicity of representative isolate, Ah01HK13 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage and inoculated by spraying spore suspension (concentration = 104 conidia/ml) to the copper-colored leaves until drops were equally distributed on it using manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed after 2 days post-inoculation (dpi) and monitored daily for symptom development (1). The experiment was repeated three times. The typical 0.1 to 0.4 mm black leaf spots were observed 7 dpi. No symptoms were observed on control plants. A fungus with the same colony and conidial morphology as A. heveae were re-isolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch's postulates. Based on these results, the disease was identified as black spot of rubber tree caused by A. heveae. To our knowledge, this is the first report of A. heveae on rubber tree in China.