Diversity Of Corynespora Cassiicola Isolates And Changes In Rubber (Hevea Brasiliensis) Leaf Protein Profiles In Response To Pathogen Inoculation

Corynespora leaf fall, caused by Corynespora cassiicola, is one of the most important diseases in rubber (Hevea brasiliensis) plantations. A study was conducted to analyse the diversity among C. cassiicola isolates and to investigate the changes in rubber leaf protein profiles in response to this pathogen. Inter Simple Sequence Repeat (ISSR) and rDNA-ITS sequence markers along with morphological characteristics and detached leaf assay were employed to analyse 21 isolates of C. cassiicola collected from different rubber clones grown in several states of Malaysia. Variations in morphological features were observed within and among isolates with no inclination to either clonal or geographical origins of the isolates. The ISSR and rDNA-ITS sequence analyses segregated the studied isolates into two distinct groups. Group 1 includes 12 isolates from the states of Johor and Selangor (this group was split into 2 subgroups 1A and 1B, subgroup 1B includes a unique isolate, CKT05D); and group 2 includes 9 isolates obtained from the other states. AMOVA analysis showed 84% of total genetic variation was attributed to variation between two groups with highly significant difference. The detached leaf assay performed on selected rubber clones grouped the isolates in subcluster 1A into Race 1; the isolates in cluster 2 into Race 2 while the pathogenicity of the isolate CKT5D was dissimilar to either Race 1 or Race 2. Two Single Nucleotide Polymorphisms (SNPs) were discovered from the rDNA-ITS region of the studied isolates. They are correlated to the races that were identified in Malaysia. The BLAST search results revealed that the nucleotide sequences in the rDNA-ITS region of C. cassiicola fungus are highly conserved. Seven SNPs and two indels were detected in the rDNA-ITS region of the studied and deposited C. cassiicola isolates obtained from several countries on diverse hosts and their presence may be correlated with the race of this fungus. The changes in the leaf protein profiles of two rubber clones RRIM 600 and PB 260 in response to inoculation with the spores of two isolates representing two races of this fungus were analysed using two-dimensional gel electrophoresis (2-DE). Several differentially expressed proteins were detected at different time points after inoculation. Dissimilarities in expression patterns were observed within and among the four clone/isolate interaction systems. The number of differentially expressed proteins was also different among the systems. These proteins differed in their estimated isoelectric points (pI) and molecular weights (MW) with the exception of three detected identical proteins. In conclusion, morphological analysis could identify but not differentiate the races of C. cassiicola; ISSR markers proved useful to distinguish the races while rDNA-ITS sequence markers could not only identify but could also infer the races of this fungus. This study confirmed that at least two distinct groups of C. cassiicola infect rubber trees in Malaysia. The changes in the 2-DE protein profiles of the rubber leaf proteomes in response to inoculation with C. cassiicola are highly dependent on the compatibility reactions of the rubber clone to a particular isolate. Differences in protein profiles implied the complexity of the interactions

Analisis Keragaman Genetik Isolat Corynespora Cassiicola (Berk & Curt) Wei. Di Indonesia Menggunakan Marker Issr (Inter Simple Sequence Repeat)

Penyakit Gugur Daun Corynespora (PGDC) yang disebabkan oleh patogen Corynespora cassiicola (Berk & Curt) Wei., merupakan salah satu penyakit penting pada tanaman karet (Hevea Brasiliensis). Klon-klon karet yang semula bersifat moderat terhadap serangan jamur ini, setelah beberapa tahun menjadi rentan dan terserang hebat. Hal tersebut diduga karena evolusi patogen menghasilkan ras baru yang lebih merusak. Oleh karena itu, informasi yang berkaitan dengan variabilitas patogen sangat diperlukan. Bahan yang dipakai dalam penelitian ini adalah isolat Corynespora cassiicola dari 7 daerah sentra perkebunan karet Indoneia yaitu Lampung, Bengkulu, Sumatera Selatan, Jambi, Sumatera Utara, Kalimantan Barat, dan Jawa Tengah. Prosedur kerja dalam penelitian ini meliputi penyiapan kultur isolat, ekstraksi DNA Corynespora cassiicola menggunakan metode modifikasi CTAB berdasarkan Situmorang (2002), analisis PCR dengan Inter Simple Sequence Repeat (ISSR), analisis data menggunakan UPGMA dalam program NTSYS, dan uji kelayuan daun menggunakan metode Nghia et al. (2007). Hasil analisis keragaman genetik isolat Corynespora cassiicola asal 7 wilayah sentra karet di Indonesia menggunakan marker ISSR menghasilkan 4 kelompok (4 ras) C. cassiicola, kelompok A (Ras 1) yaitu isolat asal Bengkulu, kelompok B (Ras 2) meliputi isolat asal Lampung dan Sumatera Utara, kelompok C (Ras 3) meliputi isolat asal Kalimantan Barat, Sumatera Selatan dan Jambi, dan kelompok D (Ras 4) yaitu isolat asal Jawa Tengah. Hasil uji kelayuan daun menunjukkan bahwa isolat-isolat Corynespora yang berada pada kelompok yang sama berdasarkan hasil analisis genetik, juga mempunyai tingkat infeksi (patogenitas) yang sama terhadap beberapa daun karet yang diuji. Isolat asal Jawa tengah (CJT) mempunyai rata-rata tingkat infeksi (patogenitas) tertinggi dibandingkan isolat yang lainnya. Tingkat infeksi terendah dari 7 isolat adalah pada klon RRIM 712 dan IRR 5, sedangkan tingkat infeksi 7 isolat Corynespora tertinggi terdapat pada klon BPM 24 dan PR 255. Diterima : 9 Juli 2012; Disetujui : 20 September 2012 How to Cite : Munir, M., Suryaningtyas, H., & Kuswanhadi. (2012). Analisis keragaman genetik isolat corynespora cassiicola (berk & curt) wei. Di Indonesia menggunakan marker ISSR (inter simple sequence repeat). Jurnal Penelitian Karet, 30(2), 86-99. Retrieved from http://ejournal.puslitkaret.co.id/index.php/jpk/article/view/12

Development and Use of a Detached Leaf Assay to Assess Soybean Cultivar Resistance to Target Spot, Caused by Corynespora cassiicola

Target spot, caused by the fungus Corynespora cassiicola, was once considered a minor foliar disease of soybean in the US. However, a severe outbreak of target spot occurred in the southeastern U.S in 2016. Due to the lack of information on resistance in soybean cultivars, and the challenges in screening cultivars in the field, a detached leaf assay was developed to evaluate resistance. The objectives of the effort were to 1) develop a greenhouse inoculation method of soybean with C. cassiicola 2) use that method to compare reactions of soybean germplasm lines to target spot 3) collect and characterize isolates of C. cassiicola from Arkansas soybean leaves. Initially, soybean seedlings were spray-inoculated with either a mycelial suspension or spores of the pathogen and placed in a dew chamber for three or more days. Small lesions developed but did not develop into typical target spot lesions. However, a detached leaf assay that placed a droplet of either inoculum on wounded leaves resulted in typical target spot lesions that continued to expand. There were significant differences between cultivars, with some cultivars showing little disease development. The detached leaf assay involved the use of detached unifoliate leaves. Wounded leaves were inoculated with a conidial suspension (1 x 105), incubated in Petri dishes on moist filter paper, and incubated at 23ºC under 12-hour light. Disease was assessed based on percent leaf area with symptoms and recorded between 7-17 days using the Bioleaf foliar analysis mobile application. Area under the disease progress curve (AUDPC) after inoculation was calculated. The assay was used to evaluate 37 breeding lines and the commercial cultivars DG48E49, LS4299XS and a moderately susceptible check, Hutcheson. Reactions ranged from resistant to susceptible and most lines were consistent between runs. A two-location field trial was conducted to determine target spot susceptibility. Although disease in both locations was low, there were significant differences between cultivars and between locations. However, there was low correlation between the field and detached leaf results. While the detached leaf assay consistently separates soybean cultivars based on disease development, further testing is required to address variability within and between tests, and additional field data is needed to determine the capability of the assay to predict field performance. To compare isolates of C. cassiicola, 28 single-spore isolates were collected from symptomatic soybean leaves at multiple Arkansas locations in 2020 and 2021. Isolates were compared morphologically in culture and the internal transcribed spacer (ITS) region of each isolate was sequenced. All isolates produced colonies and conidia consistent with C. cassiicola; isolates differed in the amount of sporulation. Based on sequence analysis in BLAST, the isolates were confirmed to be C. cassiicola with a ≥ 98% match with sequences of C. cassiicola in the National Center for Biotechnology Information (NCBI) database. The isolates were assessed for aggressiveness in the detached leaf assay by measuring the percent leaf area and calculating AUDPC. Isolate Cory 20-03 was selected to evaluate the soybean cultivars for resistance in the detached leaf assay. Despite the initial comparison of isolates showing a high level of consistency in culture and the ITS region, the variation in aggressiveness and sporulation ability suggests further sequencing of additional genes is required

Survey of cucumber target spot, in vitro sporulation and aggressiveness of Corynespora cassiicola

The objective was to carry out a survey of the occurrence of the target spot (Corynespora cassiicola) in cucumber crops in São Paulo State; to evaluate culture media for sporulation of the pathogen and the aggressiveness of isolates of the pathogen in cucumber plants. The target spot was found in nine municipalities, being the main disease in seven of the ten municipalities sampled, with leaf incidence above 50%, showing that the target spot of cucumber is widely distributed in São Paulo State. Other diseases found less frequently were scabies (Cladosporium cucumerinum), alternaria spot (Alternaria cucumerina), cercospora spot (Cercospora citrullina) and downy mildew (Pseudoperonospora cubensis), predominant in samples from one or two municipalities, but also found in most municipalities. The zoned spot (Leandria momordicae) was found to have a low incidence, in samples from three municipalities and with an incidence lower than 20%. Greater sporulation of the pathogen occurred in tomato juice and oat flour media, without scraping the surface of the colony maintained for 16 days at 25ºC, under continuous fluorescent light. The germination of C. cassiicola isolates used in the aggressiveness test was between 82.8 and 95.5%, with the 50 isolates separated into two groups. The isolates were separated into four groups within the range of 3.1 to 22.3% in severity, after ten days of inoculation of the pathogen, showing the genetic variability within the species, which should be considered in management studies, such as genetic improvement.The objective was to carry out a survey of the occurrence of the target spot (Corynespora cassiicola) in cucumber crops in São Paulo State; to evaluate culture media for sporulation of the pathogen and the aggressiveness of isolates of the pathogen in cucumber plants. The target spot was found in nine municipalities, being the main disease in seven of the ten municipalities sampled, with leaf incidence above 50%, showing that the target spot of cucumber is widely distributed in São Paulo State. Other diseases found less frequently were scabies (Cladosporium cucumerinum), alternaria spot (Alternaria cucumerina), cercospora spot (Cercospora citrullina) and downy mildew (Pseudoperonospora cubensis), predominant in samples from one or two municipalities, but also found in most municipalities. The zoned spot (Leandria momordicae) was found to have a low incidence, in samples from three municipalities and with an incidence lower than 20%. Greater sporulation of the pathogen occurred in tomato juice and oat flour media, without scraping the surface of the colony maintained for 16 days at 25ºC, under continuous fluorescent light. The germination of C. cassiicola isolates used in the aggressiveness test was between 82.8 and 95.5%, with the 50 isolates separated into two groups. The isolates were separated into four groups within the range of 3.1 to 22.3% in severity, after ten days of inoculation of the pathogen, showing the genetic variability within the species, which should be considered in management studies, such as genetic improvement

Evaluation De L’efficacité De Fongicides Au Laboratoire Contre Corynespora Cassiicola, Agent Causal De La Maladie « Corynespora Leaf Fall » De L’hévéa En Côte d’Ivoire

Corynespora leaf fall disease (CLFD) caused by Corynespora cassiicola is increasingly a major problem for the development of rubber production in Côte d'Ivoire. In the search for solutions to this problem, the efficacy of 11 fungicides (Azoxystrobin, Carbendazime + Chlorothalonil, Carbendazime, Chlorothalonil, Difenoconazole, Fosetyl-Aluminum, Iprodione, Mancozeb, Metalaxyl + Copper Oxide, Pyraclostrobin + Fenpropimorph, Triadimefon) was laboratory tested against an isolate of C. cassiicola. The results of this test showed a high level of efficacy of four fungicides, Iprodione, Carbendazime + Chlorothalonil, Carbendazim and Pyraclostrobin + Fenpropimorph. The use of these four fungicides formulations in mature plantation would be well indicated, to overcome this foliar pathology