Hybrid Necrosis of Wheat: Systematic Genetic Analysis, Fine Mapping, Distribution and Evolution in China

Hybrid necrosis of wheat is caused by two incompatible natural alleles Ne1 and Ne2 residing in normal phenotype parents. It was regarded as a barrier of gene flow between crop species. However, the necrosis alleles were still frequent in modern wheat varieties. Here, we constructed two high-density genetic maps of Ne1 and Ne2 in winter wheat. Of which, Ne1 locus was located in a span interval of 0.50 centimorgan (cM) on chromosome 5BL delimited by markers Nwu_5B_4137 and Nwu_5B_5114, while Ne2 co-segregated with markers Lseq102 and TC67744 on 2BS. Genetic allelic tests confirmed that the Ne1 allele in N9134 is different from that in Spcia and TA4152-60, while Ne2 allele in Zhoumai 22 (ZH22), Manitou, WL711 and Pan555 are also varied. The statistics substantiated that the dosage effect of Ne alleles also existed in moderate and severe hybrid-necrosis system, and the necrotic symptom may also be affected by genetic background. Furthermore, we clarified the discrete distribution and proportion of the Ne1 and Ne2 in China's major wheat regions, and concluded that introduced modern cultivars directly affect the frequencies of necrosis genes in modern Chinese cultivars (lines), especially that of Ne2. Taking investigation in spring wheat together, we proposed that hybrid necrotic alleles could positively affect breeding owing to their linked excellent genes. Additionally, based on the pedigree, we speculated the Ne1 and Ne2 in winter wheat may directly originate from wild emmer and rye, respectively. Overall, this study makes an important step toward better understanding hybrid necrosis in wheat.

In Vitro Conservation of Medicinal Plant St. John’s Wort (Hypericum perforatum L.) Through Dilution of Basic Medium

In vitro techniques could be applied for plant conservation through minimal growth. Growth reduction is generally attained by modifying the culture medium and/or the environmental condition. Conservation of St. John’s wort (Hypericum perforatum L.) in vitro through dilution of basic medium was conducted at the Tissue Culture Laboratory of Indonesian Spice and Medicinal Crops Research Institute (ISMCRI) in 2018. Sterile shoots were cultured into Murashige and Skoog (MS) medium by reducing macronutrients. The treatments used were full-MS + 0.1 mg L-1 N6 - benzyladenine (BA) as control; ¾ MS + 0.1 mg L-1 BA; ½ MS + 0.1 mgL-1 BA and ¼ MS + 0.1 mg L-1 BA. Observation of the culture growth was conducted three months after the treatments. The experiment was arranged in Completely Randomized Design with ten replications. The result showed that the use of a dilution of basic medium affected the growth of St. John’s wort. The use of ¼ MS + 0.1 mg L-1 BA composition could suppress culture growth (number of shoots, shoots length and number of leaves), without showing necrotic symptom until three months of conservation. This treatment could be used as an alternative to minimize the culture of St. John’s wort for in vitro conservation.

The Effect Of NaCl Salinity Stress To Phenolic Compound, Total Flavonoid And Antioxidant Activity Of Pegagan (Centella asiatica (L.) Urban) Leaves

Pegagan (Centella asiatica (L.) Urban) is a herbal plant that contain secondary metabolite compounds like phenol and flavonoid. NaCl salinity is one of abiotic stress that enhanced synthesis of some secondary metabolites in plants. This study was investigated the effect of NaCl salinity stress to phenolic compound, total flavonoid and antioxidant activity of pegagan leaves. Pegagan were treated with five different NaCl concentrations, 0 mM (1), 50 mM (2), 100 mM (3), 150 mM (4) and 200 mM (5) for a week. Morphological leaves were observed for the present of necrotic symptom. Phenolic compound and total flavonoid content were measured using spectrophotometer at wavelength 765 nm and 415 nm. Antioxidant activity was measured based on DPPH method. The result showed that increasing NaCl concentration cause increasing necrotic spot in leaves. Phenolic compound, total flavonoid and antioxidant activity are increased by increasing NaCl concentration. The result indicated that phenol and flavonoid have important role in plant defense mechanism against NaCl toxicity effects.

The Inhibition of Tobamovirus by Using the Extract of Banana Flower

Some extract of the banana plants parts have been identified to produce a compound that has efficacy as traditional medicine and human virus inhibitor. There was no previous report on the use of the banana flower as the plants antiviral sources for plant pathogen. The objective of this study was to identify the potency of two types of the banana flower as the Tobamovirus inhibitor substance. The antiviral was prepared from the extract of Ambon banana (Musa acuminata Colla) flower and Klutuk banana (Musa balbisiana Colla) flower, as the comparison extract of Mirabilis jalapa L. leaf which is widely known to have antiviral on various plant viruses was used. This study applied the Complete Randomized Design with three replications. Collected data includes the virus incubation period and the inhibitor power upon the local necrotic symptom on indicator plant. Results of this study showed that the extract of Ambon and Klutuk banana flower was able to inhibit the Tobamovirus with inhibition levels of 86.34% and 91.22%.IntisariBeberapa ekstrak bagian tanaman pisang diketahui memiliki kandungan senyawa yang berkhasiat sebagai obat tradisional dan zat yang dapat menghambat virus manusia. Belum pernah dilaporkan penggunaan bunga pisang sebagai sumber antiviral terhadap virus tumbuhan. Penelitian ini bertujuan untuk mengetahui potensi ekstrak bunga dua jenis pisang sebagai zat penghambat Tobamovirus. Antiviral disiapkan dari ekstrak bunga pisang Ambon (Musa acuminata Colla) dan pisang Klutuk (Musa balbisiana Colla), sebagai pembanding digunakan ekstrak daun Mirabilis jalapa L. yang sudah banyak diketahui mengandung antiviral pada berbagai virus tumbuhan. Penelitian ini dirancang menggunakan Rancangan Acak Lengkap dengan 3 ulangan. Data yang dikumpulkan meliputi masa inkubasi virus dan daya hambat gejala nekrotik lokal pada tanaman indikator. Hasil penelitian menunjukkan bahwa ekstrak bunga pisang jenis Ambon dan Klutuk mampu menghambat Tobamovirus dengan tingkat penghambatan sebesar 86,34% dan 91,22%.

Perkembangan Penyakit Diplodia pada Tiga Isolat Botryodiplodia theobromae Path dan Peran Toksin Dalam Menekan Penyakit pada Jeruk (Citrus spp.)

<p>Penyakit diplodia (Botryodiplodia theobromae) pada tanaman jeruk menyebar cukup luas di sentra jeruk Indonesia. Serangan parah penyakit dapat menyebabkan kematian apabila tidak dikendalikan. Tujuan penelitian adalah mengetahui patogenisitas dan peran toksin dari tiga isolat B. theobromae asal Pasuruan dan Magetan pada jeruk siam, pamelo, dan manis. Penelitian dilakukan di Laboratorium dan Rumah Kasa Balai Penelitian Tanaman Jeruk dan Buah Subtropika pada bulan November 2015 – Mei 2016. Penelitian terdiri atas dua percobaan, yaitu uji patogenisitas pada tanaman dan uji toksin kasar pada skala laboratorium. Uji patogenisitas menggunakan rancangan acak kelompok dengan sembilan kombinasi perlakuan terdiri atas tiga jenis isolat, yaitu Mg52A.1, dan Mg39.2 (asal Magetan), Ps8b (asal Pasuruan), serta tiga jenis tanaman jeruk (pamelo, siam, manis). Parameter pengamatan terdiri atas masa inkubasi, jumlah sampel nekrosis, dan luas gejala. Perlakuan pengujian toksin terdiri atas kontrol tanpa toksin, toksin kasar isolat Mg52A.1, toksin kasar isolat Mg39.2, dan toksin kasar isolat Ps8b. Aplikasi toksin dilakukan pada daun tiga varietas jeruk dengan rancangan acak lengkap, tiap perlakuan diulang tiga kali dan masing masing terdiri atas dua daun asal tanaman yang berbeda. Hasil penelitian menunjukkan bahwa masa inkubasi isolat Mg39.2 lebih cepat dibandingkan dengan isolat Mg52A.1 dan Ps8b. Ketiga isolat patogen B. theobromae asal Pasuruan dan Magetan memiliki patogenisitas yang sama dalam menimbulkan gejala penyakit pada jeruk pamelo, siam, dan manis, sedangkan toksin hanya berperan dalam mempercepat masa inkubasi.</p><p>Diplodia disease (Botryodiplodia theobromae) spread quite widely in Indonesia citrus center. Severe attacks of disease can cause death if it not controlled. The purpose of this study was determine the pathogenicity and the effect of toxins from three isolates of B. theobromae origin Pasuruan and Magetan on tangerine, pummelo, and sweet orange varieties. The study was conducted at Indonesian Citrus and Subtropical Research Institute during November 2015 – May 2016. This observation consisted of two experiments that pathogenicity test in screenhouse and crude toxin of patogen test in laboratory. Pathogenicity test used randomized block design arranged as factorial. The first factor was three isolates: Mg52A.1, Mg39.2 (from Magetan), Ps8b (from Pasuruan) and the second factor were kind of citrus (pummelo, tangerine , and sweet orange). The observation parameter consist of the incubation period, the number of necrotic samples and visual symptom. Crude toxin test treatment consists of a control test toxin without toxins, crude toxin Mg52A.1, crude toxin Mg39.2 toxin, crude toxin Ps8b. Application toxin carried out on the three leaf varieties of oranges. Each treatment was repeated three times and each consists of two leaves of different varieties. The results showed that the incubation period Mg39.2 isolates faster than two other isolates. Infection with different isolates and treatment of different citrus varieties shows that it did not different significantly in causing disease symptom of diplodia. Similarly result on crude toxin treatment with three isolates on three varieties showed that it were not different necrotic symptom. Thus the three isolates of pathogens B.theobromae origin from Pasuruan and Magetan have the same pathogenicity in causing disease symptoms in citrus pummelo, tangerine, and sweet orange. Toxin only play a role in accelerating the incubation period.</p>

PERANAN TOKSIN YANG DIHASILKAN OLEH BOTRYODIPLODIA THEOBROMAE DALAM MENIMBULKAN PENYAKIT DIPLODIA PADA BEBERAPA JENIS JERUK

Role of toxin produced by Botryodiplodia theobromae causes Diplodia Bark Diseases on some citrus. The purpose of the research was to study the role of toxin produced by Botryodiplodia theobromae causes diplodia bark diseases on some citrus. Research was conducted from March through November 2007. The experiment was done at the laboratory and at a glass house of the Department of Plant Pests and Diseases of the Faculty of Agriculture and the laboratory of the Faculty of Science and Mathematics Lambung Mangkurat University in Banjarbaru. For a leaf-necrosis bioassay of crude toxin production, the surfaces of the leaves were scratched near the center with a needle, and culture filtrate samples (50 µl) were placed on each wounded site. Treated leaves were incubated in a moist chamber with light at 26oC for 4 days, and toxin activity was determined by induction of veinal necrosis on the seven susceptible cultivar of citrus. The results of the experiment showed that the B. theobromae pathogens produced the toxin. The crude toxin was bioassayed for leaf necrosis to determine their ability to produce toxin. Culture filtrates of the isolate were highly toxic only on five susceptible citrus leaves siam Banjar citrus, sweet orange, lime, kaffir lime, and sour lime, indicating that the B. theobromae can produced toxin. Pathogenicity and toxin production of B. theobromae did not differ among different cultivar. While, no necrotic symptom produces on the pummelo and sunkist. Toxin production of B. theobromae produced during spore germination.

Occurrence of Potato Tuber Necrotic Isolates of Potato virus Y in a Commercial Tobacco Field in Southern Ontario, Canada

Most strains of Potato virus Y (PVY) can infect tobacco plants (Nicotiana tabacum) and cause vein clearing followed by leaf mottling, except the PVYN strain, which induces severe vein necrosis. Some isolates within the PVYN strain also cause potato necrotic tuber ringspot disease, but these have not been reported from Canadian tobacco fields. PVYNTN isolates include European (EU) and North American (NA) types that are serologically identical to PVYN, but can be distinguished by nucleic acid-based assays and potato bioassay (1,2). Some PVY isolates, PVYN-Wi or PVYN:O, resulting from a recombination between RNA molecules of PVYN and the common strain, PVYO, are identified as PVYO in serological assays, but induce necrosis in tobacco (2). In August of 2007, two samples of tobacco (N. tabacum, unknown cultivar) leaves showing necrotic symptoms resembling those induced by PVYN, PVYNTN, or PVYN-Wi were collected from a tobacco field in southern Ontario, Canada and submitted to the Charlottetown Laboratory, Canadian Food Inspection Agency, Charlottetown, PE. Virus in both samples (PVY-204 and PVY-205) reacted with PVYN-specific antibodies 1F5 and 4E7 (3) and induced vein necrosis in tobacco (N. tabacum cv. Samsun). A multiplex reverse transcription (RT)-PCR assay (1) for the simultaneous detection and differentiation of various PVY strains amplified two fragments (181 and 452 bp) associated with EU-PVYNTN isolates. Restriction fragment length polymorphism (RFLP) analysis targeting the P1 and NIb gene (3) also indicated that PVY-204 and PVY-205 were EU-PVYNTN isolates. Known isolates of PVYO, PVYN, and NA-PVYNTN were used in all evaluations as references (3). Furthermore, the nucleotide sequences of the P1 and NIb genes of PVY-204 and PVY-205 determined by automated cycle sequencing (3) and subjected to phylogenetic analysis indicated that the nucleotide and deduced amino acid sequences of both isolates were 96 and 95% identical, respectively, to NA-PVYNTN isolates reported from Canada, but 99% identical (both nucleotide and amino acid) to EU-PVYNTN isolates from Europe and Mexico (3). Potato (cv. Yukon Gold) plants mechanically inoculated with leaf sap from tobacco (N. tabacum cv. Samsun) infected with PVY-204 and PVY-205 developed various leaf symptoms including severe local and systemic necrotic lesions, leaf wilting, and leaf death in 3 to 5 weeks postinoculation under greenhouse conditions. The infected plants recovered in 5 to 6 weeks. Potato (cv. Yukon Gold) plants inoculated with leaf sap from tobacco (N. tabacum cv. Samsun) infected with a PVYNTN isolate (HX8) (3) and healthy tobacco leaf sap were used as positive and negative controls. The number and yield of the tubers harvested from infected plants were significantly reduced (50%), and PVY-204 and PVY-205 induced typical potato tuber necrotic ringspot disease in 52.6% of the progeny tubers with an average disease index of 0.364 (C. Kerlan and K. Charlet-Ramage, EAPR Virology Meeting Proceedings, 1998). PVYNTN was detected by RT-PCR and RFLP in all necrotic tubers and 66.7% of the asymptomatic tubers. Some tubers (15.8%) harvested from the infected plants were negative in RT-PCR targeting either P1 protein gene or NIb gene and showed neither external nor internal necrotic symptom. To my knowledge, this is the first evidence of the occurrence of PVYNTN isolates in field-grown tobacco plants in Canada. References: (1) J. H. Lorenzen et al. Plant Dis. 90:935, 2006. (2) R. Singh et al. Arch Virol. 153:1, 2008. (3) H. Xu et al. Can. J. Plant Pathol. 27:125, 2005.

Veinal Necrosis Induced by Turnip mosaic virus Infection in Arabidopsis Is a Form of Defense Response Accompanying HR-Like Cell Death

In the pathosystems of Turnip mosaic virus (TuMV) with Brassicaceae crops, various symptoms, including mosaic and necrosis, are observed. We previously reported a necrosis-inducing factor TuNI in Arabidopsis thaliana, a model species. In this study, we show that the necrotic symptom induced by TuNI, observed along the veins, was actually a form of defense response accompanying a hypersensitive reaction (HR)-like cell death in the veinal area. The virus is often localized in the necrotic region. The necrotic response is associated with the production of H2O2, accumulation of salicylic acid (SA), emission of ethylene, and subsequent expression of defense-related genes. Additionally, this HR-like cell death is eased or erased by a shading treatment. These features are similar to the HR-associated resistance reaction to pathogens. However, unlike HR, two phytohormones—SA and ethylene—are involved in the necrosis induction, and both SA- and ethylene-dependent pathogenesis-related genes are activated. We concluded that the veinal necrosis induced by TuMV is regulated by a complex and unique network of at least two signaling pathways, which differs from the signal transduction for the known HR-associated resistance.

Dual Role of Desferrioxamine in Erwinia amylovora Pathogenicity

To investigate the role of iron in Erwinia amylovora pathogenicity, virulence properties of two mutants of strain CFBP 1430 isolated by insertional mutagenesis and affected in the iron transport pathway mediated by desferrioxamine (DFO) were analyzed. One mutation (dfoA∷MudIIpR13) disrupts DFO biosynthesis. The present analysis shows that this mutation affects an open reading frame that belongs to a biosynthetic gene cluster and shares identity with the alcA gene required for synthesis of the siderophore alcaligin in Bordetella spp. A second mutation (foxR∷MudIIpR13) affects the synthesis of the ferrioxamine receptor FoxR, encoded by the foxR gene, and was shown to be transcribed into a monocistronic message. Accordingly, the foxR mutant accumulates DFO in the external medium. The growth of the mutants when supplied with various iron sources was examined; it indicates that the production of DFO and the specific transport of the DFO ferric complex are required only when iron is strongly liganded. Pathogenicity was scored after inoculation of apple seedlings and after infection of apple flowers. On seedlings, the DFO biosynthetic mutant behaved like the wild-type strain while the frequency of necrotic plants caused by the receptor mutant decreased by a factor of two to five, depending on the initial inoculum. On flowers, both mutants were strongly affected in their ability to initiate a necrotic symptom and their growth was reduced by two orders of magnitude relative to the wild-type strain. However, the virulence of the dfoA mutant varied with the inoculum concentration. Unlike the foxR mutant, the dfoA mutant only weakly induced plant cell electrolyte leakage in tobacco leaf disks. The supply with exogenous DFO, only when iron free, restored the ability to induce electrolyte leakage to the dfoA mutant and increased the leakage induced by other strains. DFO alone was not an inducer. Iron-free DFO was able to protect E. amylovora cells against lethal doses of hydrogen peroxide. The main conclusion was that production of DFO in E. amylovora during pathogenesis is not only a critical function for iron acquisition, but can play a role in the oxidative burst elicited by the bacteria.

Characterization of the Molecular Mechanism of Defective Interfering RNA-Mediated Symptom Attenuation in Tombusvirus-Infected Plants

ABSTRACT Different tombusviruses were able to support the replication of either homologous or heterologous defective interfering (DI) RNAs, and those infected plants usually developed typical attenuated symptoms. However, in some helper virus-DI RNA combinations the inoculated plants were necrotized, although they contained a high level of DI RNA, suggesting that the accumulation of DI RNA and the resulting suppression of genomic RNA replication were not directly responsible for the symptom attenuation. Moreover, the 19-kDa protein product of ORF 5, which is known to play a crucial role in necrotic symptom development, accumulated at the same level in the infected plants in the presence of protective homologous DI RNA and in the presence of nonprotective heterologous DI RNA. It was also demonstrated, by chimeric helper viruses, that the ability of heterologous DI RNA to protect the virus-infected plants against systemic necrosis is determined by the 5′-proximal region of the helper virus genome. The results presented suggest that DI RNA-mediated protection did not operate via the specific inhibition of 19-kDa protein expression but, more likely, DI RNAs in protective DI-helper virus combinations specifically interacted with viral products, preventing the induction of necrotic symptoms.