<meta charset="utf8"></head><body>Inoculação rápida de folhas in vivo para triagem de resistência ao cancro do caule do choupo

<ol>
	<li>Du, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+and+seedling+selection+of+the+poplar+progenies+of+Aigeiros+section.">Genetic analysis and seedling selection of the poplar progenies of Aigeiros section.</a> Journal of Huazhong Agricultural University. 28 (5), 624-630 (2009).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Identification of canker resistance to selection poplar clones [Master's thesis]. , (2014).</li><li>Zhang, L., Zhang, H., Ou, D., Fan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+on+the+resistance+to+canker+of+some+new+leuce+hybrids+inoculated+with+Botryosphaeria+dothidea.">Investigation on the resistance to canker of some new leuce hybrids inoculated with Botryosphaeria dothidea.</a> Journal of Northwest Forestry University. 32 (6), 210-213 (2017).</li><li>Du, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+selection+of+seedling+growth+traits+in+a+poplar+hybrid+population.">Genomic selection of seedling growth traits in a poplar hybrid population.</a> For Res. 36 (6), 11-19 (2023).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+in+vivo+inoculation+method+for+introducing+tree+stem+canker+pathogens+onto+leaves:+Suitable+for+large-scale+assessment+of+resistance+in+poplar+breeding+progeny.">A rapid and efficient in vivo inoculation method for introducing tree stem canker pathogens onto leaves: Suitable for large-scale assessment of resistance in poplar breeding progeny.</a> BioRxiv. , (2024).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+canker+pathogens+trigger+carbon+starvation+by+inhibiting+carbon+metabolism+in+poplar+stems.">Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.</a> Sci Rep. 9 (1), 10111 (2019).</li><li>Xing, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+pathogens+of+canker+disease+trigger+canopy+dieback+in+poplar+saplings+by+inducing+functional+failure+of+the+phloem+and+cambium+and+carbon+starvation+in+the+xylem.">Fungal pathogens of canker disease trigger canopy dieback in poplar saplings by inducing functional failure of the phloem and cambium and carbon starvation in the xylem.</a> Physiol Mol Plant Pathol. 112, 101523 (2020).</li><li>Xing, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparisons+of+photosynthetic+response+and+characteristics+in+leaves+of+Populus+alba+var.+pyramidalis+Infected+by+the+stem+canker+pathogen+Valsa+sodida+and+Botryosphaeria+dothidea+at+early+stage.">Comparisons of photosynthetic response and characteristics in leaves of Populus alba var. pyramidalis Infected by the stem canker pathogen Valsa sodida and Botryosphaeria dothidea at early stage.</a> Scientia Silvae Sinicae. 57 (09), 121-129 (2021).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Valsa+sordida+infection+on+photosynthetic+characteristics+and+carbon-water+metabolism+in+Populus+alba+var.+pyramidalis.">Effects of Valsa sordida infection on photosynthetic characteristics and carbon-water metabolism in Populus alba var. pyramidalis.</a> For Res. 34 (05), 58-68 (2021).</li><li>Balendres, M. A., De Torres, R., Dela Cueva, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+storage+age+and+fungal+re-isolation+from+host-tissue+influence+Colletotrichum+spp.+virulence+to+pepper+fruits.">Culture storage age and fungal re-isolation from host-tissue influence Colletotrichum spp. virulence to pepper fruits.</a> J Phytopathol. 167 (9), 510-515 (2019).</li><li>Ward, K. T., Ostry, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+Septoria+musiva+and+implications+for+disease+resistance+screening+of+poplars.">Variation in Septoria musiva and implications for disease resistance screening of poplars.</a> Plant Dis. 89 (10), 1077-1082 (2005).</li><li>Asalf, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogenic+resistance+of+leaves+and+fruit,+and+how+leaf+folding+influences+the+distribution+of+powdery+mildew+on+strawberry+plants+colonized+by+Podosphaera+aphanis.">Ontogenic resistance of leaves and fruit, and how leaf folding influences the distribution of powdery mildew on strawberry plants colonized by Podosphaera aphanis.</a> Phytopathology. 104 (9), 954-963 (2014).</li><li>Dunnell, K. L., Le Boldus, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+between+Septoria+leaf+spot+and+stem+canker+resistance+in+hybrid+poplar.">The correlation between Septoria leaf spot and stem canker resistance in hybrid poplar.</a> Plant Dis. 101 (3), 464-469 (2017).</li><li>Wei, J., Huang, L., Gao, Z., Ke, X., Kang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+methods+of+apple+Valsa+canker+disease+caused+by+Valsa+ceratosperma+sensu+Kobayashi.">Laboratory evaluation methods of apple Valsa canker disease caused by Valsa ceratosperma sensu Kobayashi.</a> Acta Phytopathol Sinica. 40 (1), 14-20 (2010).</li><li>Shen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+the+effectiveness+of+three+inoculation+methods+for+Valsa+sordida+in+Populus+alba+var.+pyramidalis.">Comparative study on the effectiveness of three inoculation methods for Valsa sordida in Populus alba var. pyramidalis.</a> Biology. 13 (4), 251 (2024).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>Biosharp</td>
			<td>BS-QT-027B</td>
			<td></td>
		</tr>
		<tr>
			<td>C. chrysosperma isolate</td>
			<td>China Forestry Culture Collection Center</td>
			<td>CFCC86775</td>
			<td>Separation and preservation by our laboratory</td>
		</tr>
		<tr>
			<td>Cytospora chrysosperma</td>
			<td>Plant Physiology Laboratory, Institute of Forestry New Technology, Chinese Academy of Forestry</td>
			<td>NCBI accession number: MK994101 for rRNA-ITS and MN025273 for EF1&#945; gene&#160;&#160; CGMCC number:40575</td>
			<td>Separation and preservation by our laboratory</td>
		</tr>
		<tr>
			<td>Epson Perfection V370 Photo</td>
			<td>Epson&#160;</td>
			<td>V370</td>
			<td>Scanner; Scan the leaves into image</td>
		</tr>
		<tr>
			<td>PDA (Potato Dextrose Agar)&#160;</td>
			<td>Solarbio</td>
			<td>P8931</td>
			<td>Provide nutrition for fungal growth</td>
		</tr>
		<tr>
			<td>PE plastic film</td>
			<td></td>
			<td></td>
			<td>To fix fungi on the leaves</td>
		</tr>
		<tr>
			<td>Populus alba var. Pyramidalis</td>
			<td>Plant Physiology Laboratory, Institute of Forestry New Technology, Chinese Academy of Forestry</td>
			<td></td>
			<td>Cultivated by our laboratory</td>
		</tr>
		<tr>
			<td>SPSS</td>
			<td>IBM</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>Thermostatic incubator</td>
			<td>Shanghai Kuntian Laboratory Instrument Co., Ltd</td>
			<td>KTD-6000</td>
			<td>Provide an environment for fungal growth</td>
		</tr>
		<tr>
			<td>Tough TG-6 camera</td>
			<td>OLYMPUS</td>
			<td></td>
			<td>To take photos of diseased leaves</td>
		</tr>
	</tbody>
</table>

in vivo leaf inoculation: an alternative method to assess the disease resistance of hybrid clones in poplar breeding of stem canker disease

Poplar stem canker diseases, mainly caused by two necrotrophic pathogens, Cytospora chrysosperma (Pers.) Fr. and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not., severely threaten the development and survival of poplar plantations in the Northeast, North, and Northwest (Three-north) of China. Hybrid breeding is the most direct and efficient method to control and manage tree diseases; however, compared with the progress in breeding for high-yield, fast-growing, or other poplar hybrid clones, research on resistance breeding for poplar canker disease is scarce. Only limited studies of disease-resistance hybrid breeding have been reported1,2,3, and no canker-resistance hybrid clone has been cultivated in poplar afforestation.
The crucial step of regular hybrid breeding is the clone selection based on the phenotype acquisition of hybrid progeny. However, acquiring the acquisition of pathological phenotypes (pathotypes) is time-consuming, laborious, tiring, expensive, and experts-depending&#160;process. For woody plants, it is more challenging due to the time-, labor-, and economy-consuming nature caused by their long lifecycle, slow growth, and massive body. For example, in poplar, the canker resistance screening of hybrid progeny was conducted 5-7 years after hybridization through conventional in vitro stem inoculation methods1,2,3. Moreover, limited by this low-efficiency and high-consuming disease-resistant screening method, researchers re-selected the canker-resistant clones from a small subgroup (for example, the selected high-yield or fast-growing poplar clones), not from all hybrid progeny. Therefore, using the regular stem inoculation methods, it is not sure to identify the authentic resistant clones and can not reveal the disease-resistant diversity of the breeding progeny, thus limiting the exploration of disease-resistance-related genes or gene modules. Compared to the rapid development of poplar fast-growing/high-yield breeding, those breeding programs can obtain hybrids through phenotypic selection or even genomic selection in the first two years of breeding4, the poplar canker-resistance breeding developed slowly. The disease-resistant screening (or detection) of hybrid progeny, or the pathogen inoculation method, has become the crucial speed-limiting step in poplar canker disease-resistance breeding.
In this protocol, we introduce a novel inoculation method for poplar stem canker pathogens, in vivo leaf inoculation method. Using this method, we can quickly and efficiently test the canker disease resistance of dozens of poplar species (cultivars or clones) within 5-7 days. The validation assay illustrated that the in vivo leaf inoculation is consistent with the traditional in vitro stem inoculation on the resistance detection of stem canker disease, suggesting that the leaf inoculation method is suitable for large-scale resistance screening of poplar canker diseases, such as the whole genotype selection of resistance progeny in the breeding of stem canker disease. This method solves the pathological problem of selecting resistant offspring in the hybrid breeding of poplar canker diseases.

<meta charset="utf8"></head><body>Autor em destaque: Inoculação de folhas in vivo de alto rendimento para acelerar a triagem de resistência a doenças no melhoramento híbrido de álamo

Inoculação foliar in vivo: um método alternativo para avaliar a resistência a doenças de clones híbridos no melhoramento genético de choupo da doença do cancro do caule

The scope of this research protocol is to examine the methodology of pathogen inoculation and disease-resistant assessment methods using the poplar hybrid breeding. We are proposing the in vivo leaf inoculation method as an alternative to the traditional method with the aim of advancing the breeding of poplar stem canker disease. Using the leaf inoculation method, researchers can screen the disease resistance of large-scale hybrid clones, in the second years of hybrid lesion, realizing earlier on the sliding selections in poplar hybrid grading of stem canker disease.The most advantageous aspects of the leaf inoculation method are as high efficiency and high throughput. First, the leaf method can sharply shorten the period of disease resistance selection from five to seven years after hybridization to two years after hybridization. Second, the method can assess the disease resistance of large-scale hybrid clones within several weeks.The leaf inoculation method will advance the hybrid breeding of poplar Botryosphaeria dothidea and Cytospora chrysosperma canker in China, and the poplar hybrid breeding of Septoria canker in North America. Moreover, the method can be used to mine disease resistance genes and improve the development of poplar molecular breeding. In addition to poplar hybrid breeding of stem canker disease, the leaf method can be used to screen the disease resistance of natural or mutant poplar populations, and the susceptibility of poplar stem canker pathogens.Moreover, the method can provide pathotypes for the mining of disease resistance genes, and the genetic analysis of disease resistance.

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. &#38; de Not. are the two ...

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Research

JoVE Journal

Biology

In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

288 Views.  Chinese Academy of Forestry. The scope of this research protocol is to examine the methodology of pathogen inoculation and disease-resistant assessment methods using the poplar hybrid breeding. We are proposing the in vivo leaf inoculation method as an alternative to the traditional method with the aim of advancing the breeding of poplar stem canker disease. Using the leaf inoculation method, researchers can screen the disease resistance of large-scale hybrid clones, in the second years of hybrid lesion, realizing earlie...