Diseased Leaf Samples Research Articles

MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss's wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples.

The Goss’s bacterial wilt pathogen, Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system for C. nebraskensis (Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA of C. nebraskensis NCPPB 2581T is comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned to C. nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified as C. nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed that Sphingomonas (22.7%) and Clavibacter (21.2%) were dominant in diseased samples while Pseudomonas had only 3.5% relative abundance. In non-symptomatic leaf samples, however, Pseudomonas (20.0%) was dominant with Clavibacter at 0.08% relative abundance. This discrepancy in Pseudomonas abundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss’s bacterial wilt disease; and provides the first insight on Pseudomonas community dynamics in necrotic leaf lesions.

Phenotypic and molecular evaluation of maize (Zea may L.) genotypes under field conditions in the Volta region of Ghana

Maize streak disease (MSD) is the most devastating and destructive disease of maize (Zea maysL.) in sub-Saharan Africa (SSA). Field trials were conducted in the 2014 minor and 2015 major cropping seasons to screen 16 and 17 maize genotypes, respectively, for high yield and resistance to maize streak virus (MSV) infections. The plants were scored for disease severity at 4, 6, 8 and 10 weeks after planting (WAP) based on a 1-5 visual scale (1=No infection and 5=Very severe infection). Polymerase chain reaction (PCR) test was done to detect the presence of MSV in the diseased leaf samples in order to confirm field resistance. Both phenotypic and PCR test revealed that all the maize genotypes tested in the study were infected by MSV. There was a significant varietal effect on the incidence and severity of MSD in both the major and minor seasonal trials. Genotypes ‘Abontem’,’Aburohemaa’, ‘Akposoe’, ‘Dapango’, ‘Dorke’, ‘Etubi’, ‘Honampa’, ‘Mamaba’, ‘Obatanpa’, ‘Omankwa’ and PAN 12 showed mild disease symptoms during both major and minor cropping seasons. On the other hand, genotypes ‘Dormabin’, ‘Dzinu-Eve’, ‘Enibi’, Keta 60 and PAN 53 exhibited moderate to severe symptoms during the two cropping seasons. Incidence and severity of MSD were significantly higher in the minor season than in the major season, indicating a significant seasonal effect of MSV on the maize genotypes. The yield and yield components were observed to vary significantly among the different maize genotypes and between the cropping seasons with mean yields significantly higher in the major season than in the minor season. Genotypes ‘Abontem’, ‘Aburohemaa’, ‘Akposoe’, ‘Dorke’, ‘Etubi’, ‘Honampa’, ‘Omankwa’, ‘Obatanpa’ and PAN 12 (All improved varieties), which exhibited partial resistance to MSV infection gave high seed yields during both seasons. The improved maize genotypes that were high yielding and resistant to MSV infection should be evaluated for uniform yield trials on farmers’ fields towards their release as varieties to farmers.

Development and application of a LAMP method for rapid detection of apple blotch caused by Marssonina coronaria

Marssonina coronaria is a devastating plant pathogen that causes apple blotch on apple. Early diagnosis of M. coronaria is the key to control apple blotch disease effectively. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of M. coronaria. The ribosomal DNA internal transcribed spacer (rDNA-ITS) sequence of M. coronaria was selected as the target for primer design. The LAMP reaction was optimal at 60 °C for 70 min based on the color change and gel electrophoresis. In specificity tests, the LAMP assay was able to distinguish M. coronaria from other apple pathogenic fungi. In sensitivity tests, the detection limit of the LAMP assay was 100 fg μL−1 genomic DNA of M. coronaria. Furthermore, the LAMP assay was successfully applied to detect M. coronaria in diseased apple leaf samples from the field. Thus, our study provides a simple and efficient method for quick diagnosis of apple blotch caused by M. coronaria.

First Report of Damping-off on Sedum plumbizincicola Caused by Rhizoctonia solani AG 2-1 in China.

Sedum plumbizincicola X.H. Guo et S.B. Zhou sp. nov. is a plant species of the family Crassulaceae that has the ability to hyperaccumulate cadmium and zinc in high concentrations (Liu et al. 2017). In April of 2018 and 2019, a disease of damping-off was observed on S. plumbizincicola seedlings in a nursery in Changsha (28°13'N; 112°56'E), the Hunan Province of China, in which nearly 1 million seedlings were planted. Approximately 40% of the surveyed plants were infected. The affected plants displayed water-soaking on the shoots and stems, and chlorosis on the leaves. As the disease spread upward, leaf stalks or the whole plants became wilted and collapsed. Five diseased stem and leaf samples were collected. Symptomatic tissues were excised and surface sterilized with 70% ethanol for 10 s, and 0.1% HgCl2 for 2 min, washed with sterile distilled water for three times, and then cultured on potato dextrose agar (PDA) at 26°C in darkness. Fungal colonies were similar in morphology: white, light gray to brown, with hyphae branched at nearly right angles, septa near the branching point and constrictions at the base of hyphal branches. After 10 days, white-gray to brown sclerotia were produced. The morphological characteristics were consistent with those of Rhizoctonia solani J.G. Kühn (Sneh et al. 1991). Genomic DNA of a representative isolate was extracted using the cetyltrimethylammonium bromide method. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced with the primer pairs ITS4/ITS5 (White et al. 1990). When analyzed by the BLASTn program, the ITS sequence (GenBank Accession No. MN961664) had 100% identity to the corresponding gene sequence of R. solani anastomosis group (AG) 2-1(Accession Nos: LC202869.1 and MH862641.1). In addition, primers Rhsp1/ITS4B and Rhsp2/ITS1F specific for R. solani, and AG21sp/ITS4B specific for R. solani AG 2-1 were also used (Salazar et al. 2000). Results revealed that our isolate was R. solani AG 2-1. Pathogenicity was confirmed via in vivo inoculation of one-month-old S. plumbizincicola seedlings in sterilized nursery soil with four representative isolates. For each pot, five 5-mm-diameter mycelial plugs from 7-days old colonies on PDA were placed in the soil near the base of the stems. Plants inoculated with agar plugs without mycelium served as controls. The inoculated plants were kept in a growth chamber at 25°C with a 12/12 h light/dark cycle. Pathogenicity tests were performed twice, with three replicative potted plants for each isolate in each test. Approximately 25 days after inoculation, the damping-off symptoms resembling those observed in the field were displayed on the inoculated plants, while no obvious symptoms were observed on the control plants. R. solani was re-isolated from all infected plants and molecularly characterized, thus confirming Koch's postulates. R. solani has been previously reported as the pathogen of damping-off disease in many plants, such as canola (Paulitz et al. 2006) and oat (Zhang et al. 2016). However, to the best of our knowledge, this is the first report of R. solani causing damping-off of S. plumbizincicola in China. S. plumbizincicola is widely planted for heavy metal pollution treatment in China. The occurrence of this disease could seriously affect the production of the seedlings, and management strategies should be developed.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Canna edulis Ker in China.

Canna edulis Ker has been an important economic plant in southern China. The tuberous stems are the most valued plant part and in the past were routinely used as animal feeds. In June 2019, leaf spot disease were detected on C. edulis plants in Chengmai, Hainan Province. Symptoms of the disease were characterized by oval-shaped, initially pale to yellow lesions that become necrotic (brown) with yellow borders, As the lesions expanded, the disease could encompass the entire leaves. which were seen as concentric rings typical of anthracnose disease (FigS1-A). A survey of C. edulis plants revealed that the disease caused serious damage during the summer in Hainan Province, with 50 to 60% incidence in plants. To isolate the pathogen, ten pieces of diseased leaf samples were plated and seven pieces yielded fungal colonies after 5 to 6 days of incubation at 25 °C. The Colonies were single-spored to obtain pure cultures. Pure cultures on potato dextrose agar (PDA) appear white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown (FigS1-B). Conidia were single-celled, hyaline, cylindrical to slightly curved with a rounded apex and truncated base that measured 13.3 to 18.1(length) × 3.7 to 5.5 (width) μm (n=50) (FigS1-C). The morphological characteristics and measurements of this fungal pathogen matched the previous descriptions of Colletotrichum gloeosporioides (Prihastuti et al. 2012). Isolate JO-3 was identicated by molecular analysis, sequences of the internal transcribed spacer (ITS), actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions were obtained. These loci were amplified from isolates using the primer pairs ITS1/ITS4 (Mills et al. 1992), ACT-512F/ ACT-783R (Weir et al. 2012) and GDF/GDR (Templeton et al. 1992) respectively. A BLAST search of GenBank showed that the ITS (MN913584), ACT(MN919196) and GAPDH (MN919195) sequences of the isolate were 99% , 100% and 100% homologous with C. gloeosporioides (GenBank accession nos. MH930419, JX009931 and KX885158). Maximum likelihood trees based on concatenated sequences of the three genes were constructed using MEGA7.0. The results showed the strains isolated from C. edulis were closely related to C. gloeosporioides, as supported by high bootstrap values (FigS1-D). Pathogenicity test was performed with isolate JO-3 by depositing 10-µl droplets of a suspension (1 × 106 conidia/ml) on the surfaces of five artificially wounded leaves (a small hole made with a pushpin) of ten healthy 1-year-old C. edulis plants. An equal number of control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C with 80% humidity and observed for symptom every day. One weeks after inoculation, all the inoculated plants showed symptoms of yellow sunken spots similar to those observed in the field. No symptoms were observed on the controls. The fungus re-isolated from the infected tissues showed the same cultural and morphological characteristics of the strain inoculated, fulfilling Koch's postulates. C. gloeosporioides was previously reported as the causal agent of anthracnose on Hymenocallis littoralis (Zhao et al. 2019), Abelmoschus esculentus (L.) Moench (Shi et al. 2019) and Sorbaria sorbifolia (Li et al. 2019) in China. To our knowledge, this is the first report of anthracnose on C. edulis caused by C. gloeosporioides in China. This disease can seriously affect the yield and quality of C. edulis in China. Given its wide host range, C. gloeosporioides has great potential to become an economically important plant pathogen. The project was partially funded by Hainan Provincial Research Institute of technology development projects (Collection, Evaluation and Domestication Cultivation of Wild Vegetable Germplasm Resources in Hainan), Hainan Provincial Key Laboratory for Vegetables and Biology，Hainan Provincial Engineering Research Center for Melon and Vegetable Breeding, Major scientific and technological projects in Hainan Province(ZDKJ2017001)，Key R & D projects in Hainan Province (ZDYF2019066), The third Survey and Collection of Crop Germplasm Resources in China.

English

Fruit of Allanblackia floribunda is an active ingredient in the pharmaceutical industry. Fruit production in the species is hindered by incidence of pathogenic fungi leading to economic loss. This study was conducted to investigate micro-fungi species associated with leaf spot of A. floribunda. Field surveys were carried out in natural stands containing matured A. floribunda trees located at Owu Ikija, Ogun State (6.80°N, 4.03°E) and Benin, Edo State (6.29°N and 5.58°E) in Southern Nigeria. Diseased leaf samples were collected during wet and dry seasons. Pure cultures of fungal isolates obtained from the leaf samples were examined to determine their cultural and morphological characteristics. Percentage incidence of micro-fungi in each location was estimated. Leaves of healthy seedlings were sprayed with 104 conidial/ml spore concentration of fungal isolates to determine their pathogenicity. Fourteen fungal species were isolated from leaves of A. floribunda across the two sites. Aspergillus spp., Macrophomina phaseolina, Penicillium species, Pestalotia palmarum, Rhizopus nigricans and Trichoderma pseudokoningii were isolated from both sites during both seasons. Fusarium oxysporum, Lasiodiplodia theobromae, Penicillium javanicum and Pythium aphanidermatum were present at Owu Ikija while Colletotrichum capsici, C. coccodes and Curvularia lunata were present at Benin with fungal incidence of 12.5, 12.5 and 4.17%, respectively. P. palmarum had modal fungal incidence (35.29%) at Benin followed by T. pseudokoningii at Owu-Ikija with frequency value of 18.75 and 17.54%, respectively. P. palmarum was the most prevalent out of all micro-fungi species associated with A. floribunda in all locations. Pathogenicity test was negative for all tested isolates, variety of micro-fungi are associated with A. floribunda. Key words: Allanblackia floribunda, fruit, fungal isolates, leaf samples, pathogenicity.

First Record of Leaf Spot Disease on Costus speciosus Caused by Nigrospora oryzae in Hainan, China.

Costus speciosus (Koen.) Smith has been an important medicinal agent in the various traditional and folk systems of medicine in southern China. In September 2018, leaf spot disease was detected on C. speciosus plants in Chengmai County, Hainan Province. A survey of C. speciosus plants revealed that the disease caused serious damage during the typhoon season of September to November in Hainan Province, with 80 to85% incidence in plants. Early symptoms were yellow-to-brown, irregular-shaped lesions on the leaf margin or tip. After several days, lesions expanded along the mid-vein until the entire leaf was destroyed. Then, the infected leaves turned gray brown, leading to defoliation. Heavily infected leaves became dry and died. The pathogen was consistently isolated from the lesions and pure single-spore cultures were obtained. Twenty pieces of diseased leaf samples were plated and seven pieces yielded fungal colonies after 5 to 6 days of incubation at 25 °C. Colonies on potato dextrose agar (PDA) were white and later became gray to black. Conidia were unicellular, terminal, black, elliptical that measured 10 to 13 (length) × 12 to 16 (width) μm (n=30), growing aerial mycelium covering the entire petri dish (9 cm in diameter). The morphological characteristics and measurements of this fungal pathogen matched the previous descriptions of Nigrospora oryzae (Wang et al. 2017). To confirm identity the internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using primers ITS1/ITS4 (Mills, P. R., et al. 1992), and the 530-bp product (GenBank Accession No. MK280694) of the ITS showed 99% sequence identity to N. oryzae isolates TLFa21 (GenBank Accession No. MH790146) and xsd08022 (GenBank Accession No. EU918714). Pathogenicity tests were conducted. Three leaves of three C. speciosus plants were wounded and inoculated with mycelial plugs (5×5mm) , and an additional five plants were inoculated with PDA plugs as a control. All plants were placed in the field and temperature ranged from 23 to 29°C. Ten days after inoculation, all the inoculated plants showed typical leaf spot symptoms, a yellow-to-brown mildew appeared at the points of inoculation. No symptoms were observed on the controls. The fungus was re-isolated from the infected tissues, fulfilling Koch's postulates. N. oryzae was previously reported as the causal agent of leaf spot on cotton (Zhang. et al. 2012), dendrobium candidum (Wu. et al. 2014) and Aloe vera (Zhai. et al. 2013) in china. To our knowledge, this is the first report of leaf spot of C. speciosus caused by N. oryzae in China. The project was partially founded by Hainan Provincial Research Institute of technology development projects (Screening and application of endophytic bacteria with high resistance to Fusarium Wilt of Sauropus androgynus), Hainan Provincial Key Laboratory for Vegetables and Biology，Hainan Provincial Engineering Research Center for Melon and Vegetable Breeding, Major scientific and technological projects in Hainan Province(ZDKJ2017001)，Third Survey and Collection of Crop Germplasm Resources in China, Collection, identification and preservation of pathogenic bacteria of inverted season vegetable in Hainan.

PENYEBAB DAN SERANGGA VEKTOR PENYAKIT KERITING PADA TANAMAN LADA

The cause of stunting disease of black pepper (Piper nigrum) in Indonesia has not yet been confirmed cither due o a virus or mycoplaam. However, similar disease found on black pepper plants in the Southeast Asia is caused by Piper yellow mottle virus (PYMV) which is transmitted by Planococcus citri. This expeiments was aimed o examine the cause of the stunting disease and its insect vectors. The expeiment were conducted from October 1998 to May 2001. Diseased vegetative materials of black pepper plants showing stunting disease were collected from Sukamulya, Sukabumi. The potential insect vectors were collected from black pepper plants in Bogor, IP Sukamulya (Sukabumi), Lampung and Bangka. The insects were fed on the diseased plants obtained from Lampung, Bogor and IP Sukamulya, then transferred o the healthy plants. The healthy plants were produced rom true seeds and cutings oiginated rom Bogor and IP Sukamulya. The tested plants were incubated at the green house and examined for disease development. Diseased leaf samples collected rom black pepper plant rom IP. Sukamulya and the transmitted plants were sent to the University of Minnesota, USA for Ihe virus (PYMV) by using ISEM (immunosorbent electron microscope). The result snowed that the leaves samples rom IP. Sukamulya were infected by PYMV. The morphology and size of the virus were similar to those caused stunting disease in the South East Asia. Potential insects vectors found on the diseased black <br /><br />pepper plants were two mealybugs, P. minor and Ferrisia virgata (Hemiptera; Coccoidca: PaaidbcoccidaeX aa well as an aphid Toxoptera aurantii (Hemiptera: Aphidoidea: Aphididae). The first tial on disease transmission by using P. minor, previously reared on the potato tubers, showed one out of ten tested plants produced disease sympom. The subsequent trial using P minor, bred on healthy black pepper seedlings, showed thee out often tested plants developed disease symptoms. None of the aphid transmitted plants developed Ihe disease. This study confirmed thai PYMV b the cause of stunting disease on black pepper in IP Sukamulya and Lampung and P. minor as the insect vecor of the disease.<br /><br />

Penyebab Dan Serangga Vektor Penyakit Keriting Pada Tanaman Lada

The cause of stunting disease of black pepper (Piper nigrum) in Indonesia has not yet been confirmed cither due o a virus or mycoplaam. However, similar disease found on black pepper plants in the Southeast Asia is caused by Piper yellow mottle virus (PYMV) which is transmitted by Planococcus citri. This expeiments was aimed o examine the cause of the stunting disease and its insect vectors. The expeiment were conducted from October 1998 to May 2001. Diseased vegetative materials of black pepper plants showing stunting disease were collected from Sukamulya, Sukabumi. The potential insect vectors were collected from black pepper plants in Bogor, IP Sukamulya (Sukabumi), Lampung and Bangka. The insects were fed on the diseased plants obtained from Lampung, Bogor and IP Sukamulya, then transferred o the healthy plants. The healthy plants were produced rom true seeds and cutings oiginated rom Bogor and IP Sukamulya. The tested plants were incubated at the green house and examined for disease development. Diseased leaf samples collected rom black pepper plant rom IP. Sukamulya and the transmitted plants were sent to the University of Minnesota, USA for Ihe virus (PYMV) by using ISEM (immunosorbent electron microscope). The result snowed that the leaves samples rom IP. Sukamulya were infected by PYMV. The morphology and size of the virus were similar to those caused stunting disease in the South East Asia. Potential insects vectors found on the diseased black pepper plants were two mealybugs, P. minor and Ferrisia virgata (Hemiptera; Coccoidca: PaaidbcoccidaeX aa well as an aphid Toxoptera aurantii (Hemiptera: Aphidoidea: Aphididae). The first tial on disease transmission by using P. minor, previously reared on the potato tubers, showed one out of ten tested plants produced disease sympom. The subsequent trial using P minor, bred on healthy black pepper seedlings, showed thee out often tested plants developed disease symptoms. None of the aphid transmitted plants developed Ihe disease. This study confirmed thai PYMV b the cause of stunting disease on black pepper in IP Sukamulya and Lampung and P. minor as the insect vecor of the disease.

The biochemical changes in cluster bean leaves due to Alternaria blight infection

Cluster bean is being grown in India since ancient time. Although believed to be of African origin (Vavilov, 1951), it was domesticated centuries ago in the north-western region of the Indo Pakistan sub-continent (Hymowitz, 1972). Tender green guar pods are important source of nutrition to human being and animals. Alternaria blight is an important disease as it acts as one of the limiting factors in its production. The present investigations and studies were carried on variability of pathogen and its disease management. The biochemical data indicate that the content of protein, total soluble sugar, reducing sugar and chlorophyll in diseased leaf of different varieties gradually and significantly decreased as compared to healthy leaf of same variety. In contrast the total phenol and O-dihydroxy phenol content were significantly increased in diseased leaf samples with the progress of the disease as compared to healthy leaf samples. Protein, total sugar, reducing sugar and chlorophyll contents were decreased with the infection of pathogen in the diseased sample whereas the total phenol and O-dihydroxy phenol were increased with increasing the disease in the diseased leaf sample of different varieties.

Screening of Cowpea (Vigna unguiculata ( L.)Walp) Varieties for Resistance to Leaf Spot in Southern Guinea Savannah Agro- Ecology of Nigeria

Five cowpea varieties (Vigna unguiculata (L.) Walp) namely UAM 09 1055-6, UAM 09 1051-1, IT 99k-573-1-1, IT 90k-277-2 and IT 99k-573-2-1 were investigated for fungi associated with the seed, leaf spot disease incidence and severity in Makurdi (07o 45’- 7o 50’N and 08o 45’ - 08o 50’E ; 98 m)and Otobi (7°07’ - 7°11'N and 8° 05- 8°10'E) in Benue State, Nigeria under natural infection. The effect ofleaf spot incidence and severity on the growth and yield of cowpea varieties were also evaluated. Fungi associated with the seeds and leaf spot lesions were isolated and identified. Diseased leaf samples from the study locations were found to be infected with Fusarium verticillioides, Curvularia lunata, Aspergillus tamarii Kite, Lasiodiplodia theobromae, Aspergillus flavus Link and Aspergillus niger van Tiegh, Pythium spp, Fusarium solani, Macrophomina phaseolina and Phoma sp. Otobi field had significantly higher incidence and severity of leaf spot disease than the Makurdi field. Cowpea variety IT 99k-573-1-1, IT 99k-573-2-1 and IT 90k-277-2 were classified as moderately resistant in Makurdi with a mean incidence of 22.23%, 16.97% and 16.67% respectively while varieties UAM 09 1051-1 and UAM 09 1055-6 were classified as Moderately susceptible and Highly susceptible with mean leaf spot incidence of 41.67% and 99.17% respectively. In Otobi, all the cowpea varieties screened were classified as Susceptible to leaf spot incidence with the exception of variety IT 90k-277-2 which was classified as moderately susceptible to leaf spot disease.
 Key words: Cowpea, leaf spot, screening, resistant, susceptible.

New record of rust disease caused by Puccinia oxalidis on Oxalis latifolia from India

A severe rust infection was observed on the plantations of Oxalis corniculata (Oxalidaceae), commonly known as procumbent yellow sorrel, in Himachal Pradesh and Chandigarh, and on O. latifolia, known as garden pink-sorrel, in Uttarakhand in northern India. Detailed morphological examination of the diseased leaf samples was conducted, which confirmed the identity of the pathogen as Puccinia oxalidis. Rust symptoms on the host plants, along with taxonomic account of the phytopathogen are detailed in this paper. A taxonomic key of Puccinia species reported from Oxalis species is provided to facilitate its identity. In addition to understand its global host range, a worldwide host distribution of P. oxalidis is provided. The present study is the first detailed taxonomic account of P. oxalidis on Oxalis corniculata from Himachal Pradesh and Chandigarh in northern India. To the best of our knowledge, this is a new record of P. oxalidis from O. latifolia from India.

Qualitative Resistance of Sarawak Rice Landraces Against Pyricularia oryzae

Malaysia rice production is threatened by rice blast disease, caused by Pyricularia oryzae. Yield can be greatly reduced by this disease as it can attack all the aerial parts of rice including leaves, node, neck, and collar. The use of resistant cultivar, which can be produced from resistance breeding, can control the disease effectively. Sarawak, in Malaysian Borneo, has diverse rice landraces, which can be genetic resources for resistance breeding. Study on the resistance of Sarawak rice landraces against P. oryzae, is still limited. In this study, diseased leaf samples were collected from rice fields in Serian division, Sarawak. One isolate was successfully obtained and designated as B2PG. The morphological characteristics were documented. Six Sarawak rice landraces were challenged with isolate B2PG. Four of the rice landraces were resistant and might carry resistance gene(s), which can be utilised in future breeding program.

First Report of Leaf Spot Caused by Stemphylium lycopersici on Zinnia elegans in China

Zinnia elegans originated from Mexico and has been widely cultivated as an ornamental plant in China. In August 2018, leaf spots were observed on Z. elegans in Tai’an, Shandong province, China. The symptoms appeared on the edge of the leaves as small brown spots. In serious cases, the leaves were withered, which affected the ornamental value of the plants. Diseased leaf samples were cut into small segments, put onto moist filter papers within Petri dishes, and incubated at 25°C. Spores resembling Stemphylium developed from the tissues after 1 day and were singly picked up and transferred to potato dextrose agar (PDA). Morphologically, all the colonies were identical to each other, and the strain YZU 181216 was selected for further study. A pure culture of YZU 181216 was deposited in the Fungi Herbarium of Yangtze University, Jingzhou, Hubei, China. To determine the colony morphology of the species, mycelial plugs (6 mm in diameter) were transferred to fresh PDA and incubated at 23°C in darkness. After 7 days, the colony was vinaceous buff, reverse cinnamon to buff, 52 to 53 mm in size. To examine its conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C under an 8-h photoperiod for 7 days. On PCA, conidiophores were straight or curved, 88 to 179 × 3.5 to 6 µm in size; conidia were solitary, oblong, three to five transverse septa, 24 to 59 × 11.5 to 20.5 µm in size, with a conical apex and bluntly rounded base. Based on morphology, the fungus was identified as Stemphylium lycopersici (21 to 60 × 12 to 24 µm) described by Yamamoto (1960). To corroborate morphological identification, genomic DNA of the strain was extracted from 3-day-old mycelia grown on PDA. Three gene fragments, including the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (gapdh), and calmodulin (cmdA), were amplified with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences of the strain were deposited in the GenBank with accession numbers MK895975 (ITS), MK895977 (gapdh), and MK895976 (cmdA). Relevant gene sequences of Stemphylium spp. were derived from GenBank, and the three gene sequences were combined after alignment. Maximum likelihood analysis based on the combined three gene sequences was conducted with an evolutionary model of GTR+I+G under 1,000 bootstrap replicates. The phylogenetic tree showed that the strain YZU 181216 clustered with S. lycopersici (CBS 124980 and CBS 124981) and was supported by a bootstrap value of 100%. To confirm its pathogenicity, mycelial plugs (6 mm in diameter) were inoculated on living leaves of Z. elegans. Control groups were inoculated with fresh PDA plugs. The plants were covered with plastic bags and incubated in a greenhouse at 25°C. Brown spots were observed on the leaves after 2 days. Infected tissues withered after 5 days, similar to the original symptoms that occurred in the field. No symptoms were observed on control groups. The fungus reisolated from infected leaves was identical to S. lycopersici based on conidial morphology and gapdh gene sequence. To our knowledge, this is the first report of Z. elegans leaf spot caused by S. lycopersici in China.

Soybean leaf disease detection and severity measurement using multiclass SVM and KNN classifier

Soybean fungal diseases such as Blight, Frogeye leaf spot and Brown Spot are a significant threat to soybean plant due to the severe symptoms and lack of treatments. Traditional diagnosis of the thease diseases relies on disease symptom identification based on neaked eye observation by pathalogiest, which can lead to a high rate of false-recognition. This work present a novel system, utilizing multiclass support vector machine and KNN classifiers, for detection and classification of soybean diseases using color images of diseased leaf samples. Images of healthy and diseased leaves affected by Blight, Frogeye leaf spot and Brown Spot were acquired by a digital camera. The acquired images are preprocessed using image enhancement techniques. The background of each image was removed by a thresholding method and the Region of Interest (ROI) is obtained. Color-based segmentation technique based on K-means clustering is applied to the region of interest for partitioning the diseased region. The severity of disease is estimated by quantifying a number of pixels in the diseased region and in total leaf region. Different color features of segmented diseased leaf region were extracted using RGB color space and texture features were extracted using Gray Level Co-occurrence Matrix (GLCM) to compose a feature database. Finally, the support vector machine (SVM) and K-Nearest Negbiour (KNN) classifiers are used for classifying the disease. This proposed classifers system is capable to classify the types of blight, brown spot, frogeye leaf spot diseases and Healthy samples with an accuracy of 87.3% and 83.6 % are achieved.

Computer Aided Classification and Detection of Leaf Disease using ANN

The contribution of a plant is highly important for both human life and environment. Diseases will affect plant, like all humans and animals. Various diseases may affect plant which disturbs the plants normal growth. Leaf, stem, fruit, root, and flower of the plant may get affected by these diseases. Without proper care the plant may die or its leaves, flowers, and fruits drop. Finding of such infections is required for exact distinguishing proof and treatment of plant sicknesses. The current technique for plant malady discovery utilizes human contribution for distinguishing proof and characterization of illnesses and these strategies endure with time-unpredictability. PC supported programmed division of illnesses from plant leaf utilizing delicate registering can be fundamentally valuable than the current techniques. In this paper, we proposed a method using Artificial neural network (ANN) for identification, classification and segmentation of diseases in plant leaf automatically. In the proposed system capturing the leaf images is done first and then contrast of the image is improved by using Contrast Limited Adaptive Histogram Equalization(CLAHE) method. Then, color and texture features are extracted from the segmented outputs and the ANN classifier is then trained by using that features and it could able to separate the healthy and diseased leaf samples properly. Exploratory outcomes demonstrate that the arrangement execution by ANN taking list of capabilities is better with an exactness of 98%.

Prevalence and polymerase chain reaction detection of <i>Xanthomonas axonopodis</i> pv. <i>manihotis</i> causal agent of cassava bacterial blight disease in Osun state, Southwestern Nigeria

Bacterial blight is one of the major diseases of cassava, caused by Xanthomonas axonopodis pv. manihotis (Xam) Bondar (Vauterin et al.) in Nigeria. It induces extensive necrosis on the leaves, twigs and stems systemically, which accounts for significant yield loss. Field survey for symptoms of cassava bacterial blight (CBB) disease was conducted across ten Local Government Areas (LGAs) notable for cassava production in Osun State. Disease incidence and severity were determined by visual examination and scoring of infected plants. Leaf samples obtained from 60 farmers' fields spread over the ten LGAs during the rainy season of 2016 were bioassayed using polymerase chain reaction (PCR) to detect the presence of the pathogen. Two Xam specific primers, 1525R (5'-AGGAGGTGATCCAGCC-3'). and 27F (5'-AGAGTTTGATC (A/C)TGGCTCA-3') were used to amplify 16s gene in total nucleic acid extracted from diseased leaf samples. Leaf samples from Ife North LGA had the highest incidence and severity of 76.10% and 4.5 respectively of CBB disease. PCR results showed high DNA concentration and presence of Xam in eight LGAs with infectivity varying from mild to highly infected. However, further studies will be required to determine the diversity among Xam isolates in the State in order to elicit appropriate control measures. Keywords: Bioassay, Disease severity, Germplasm, Polymerase chain reaction, Xanthomonas axonopodis pv. manihotis

Leaf rot caused by Rhizopus oryzae on pak choy (Brassica campestris ssp. chinensis) in China.

Pak choy ( Brassica campestris ssp. chinensis L.) is the major vegetable crop cultivated in China. During December 2017, leaf rot was frequently observed on leaves of pak choy in greenhouses of Shanghai, China. Diseased leaf samples were plated onto acidified potato dextrose agar and fungal cultures were isolated and identified as Rhizopus oryzae , based on morphological features and molecular identification. Definitive identification as R. oryzae was based on the comparative molecular analysis of rRNA gene sequences. Blast analysis revealed 99% similarity with R. oryzae . Pathogenicity was determined by inoculating healthy pak choy leaves and plants with hyphal suspensions of R . oryzae. The fungus was re-isolated from developing similar lesions on the inoculated plants and identified as similar to the inoculated fungus, thus fulfilling Koch’s postulates. This is the first report of fungal leaf rot caused by R. oryzae on pak choy in China.

First Report of the Occurrence of Bacterial Leaf Streak of Corn Caused by Xanthomonas vasicola pv. vasculorum in Brazil

Bacterial leaf streak of corn, caused by Xanthomonas vasicola pv. vasculorum, was recently reported in the United States and Argentina (Korus et al. 2017; Lang et al. 2017; Plazas et al. 2018). Further, the disease has reached epidemic levels in some corn-growing states of the United States (Korus et al. 2017; Lang et al. 2017). In April 2018, diseased corn leaf samples were collected in commercial fields in the western region of the state of Parana, Brazil. The leaves with initial symptoms of the disease had translucent yellow to brown narrow linear lesions with irregular margins, confined to the interveinal spaces. Older symptoms on highly susceptible corn hybrids had coalescing long lesions of dark coloration, reaching large extension along the leaf blade. Preliminary examinations did not show any structure of fungi but revealed the presence of bacterial ooze. Bacterial isolates established on nutrient agar medium produced yellow mucoid colonies typical of bacteria of the genus Xanthomonas. The pathogenicity of the bacterial isolates was confirmed by spray inoculation of a 10⁸ CFU/ml suspension in plants at the V4 stage of the Zea mays hybrid JM 2M77, kept under greenhouse conditions. Lesions similar to those observed in the samples from commercial corn fields started to show up 9 days after inoculation. We reisolated consistently from the affected leaf tissues bacteria identical to those originally inoculated. The bacterium obtained had a rod-shaped cell, was gram negative, nonfluorescent and nonfermentative. Based on the analysis of polymerase chain reaction amplified sequences of the 16S rDNA region, using the universal primers fD1 and rD1 (Weisburg et al. 1991), the bacterial isolates showed genetic similarity of 99% with X. vasicola pv. vasculorum strain SAM119 (NCCPB no. 4614), isolated from corn in South Africa, the pathotype strain of the pathovar (Lang et al. 2017). Further, a neighbor-joining phylogenetic tree reconstructed based on partial 16S rDNA sequences alignment using MEGA7 (Kumar et al. 2016) confirmed that our bacterial strains clustered with X. vasicola pv. vasculorum strain SAM119. The sequences of two bacterial strains established in our studies were deposited in GenBank as accessions MH497007 and MH497008. The disease was present in commercial fields in at least eight municipalities of corn-producing regions of the state of Parana, Brazil. In addition, bacterial leaf streak of corn was detected under natural conditions on Z. mays plants of at least 30 different commercial hybrids. Although the occurrence of X. vasicola pv. vasculorum causing bacterial leaf streak of corn has been suspected in Brazil for previous years, this is the first confirmed report.

English

Viral infections are a major challenge to sustainable cowpea production in sub-Saharan Africa (SSA). Nine cowpea (Vigna unguiculata L. Walp) genotypes were evaluated for resistance against viral infection in a field trial involving randomized complete block design with 4 replications. Viral disease severity was assessed at 2, 4 and 6 weeks after planting (WAP) based on disease symptoms. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) using antisera raised against cucumber mosaic virus (CMV), cowpea severe mottle virus (CPSMV), cowpea mosaic virus (CPMV) and southern bean mosaic virus (SBMV) was used to detect the viruses associated with diseased leaf samples collected from the field. Biometric and yield data were also taken. The mean disease incidence and area under disease progress curve (AUDPC) varied significantly (P < 0.05) among the cowpea genotypes, with 7 (Apagbaala, UCC-366, UCC-489, UCC-490, UCC-497, UCC-514 and UCC-523) out of 9 cowpea genotypes showing field resistance whilst the other two (UCC-473 and UCC-484) exhibited moderate resistance. ELISA showed that all the 9 cowpea genotypes were infected with at least one of the three viruses namely CMV, CPMV and CPSMV, whereas SBMV was not detected. Co-infection by CMV, CPMV and CPSMV was observed in UCC-366. Mean plant height, canopy diameter and seed yield differed significantly (P < 0.05) among the cowpeas. UCC-473, UCC-316, and UCC-523 had high mean seed yields of 6.690, 4.922 and 4.144 t ha-1 respectively, above the overall mean seed yield of 3.63 t ha-1, emphasizing their resilient to viruses. Key words: Vigna unguiculata, viral infection, disease incidence and severity, resistance screening.