Resistance of Microbial Community of Artemisia annua L.to Pathogenic Fungi

[Objectives]This paper was to figure out whether the dominant bacterial community has the role and effect of bacterial community and its defense mechanism against potential pathogenic fungi in Artemisia annua,and thus establish a systematic model of bacteria-fungus-plant.[Methods]Fifty-eight strains of bacteria and one strain of pathogenic fungi,Globisporangium ultimatum,were used for the experiments.These 58 bacterial strains were assembled into a bacterial community,and the bacteria with abundance in the top 1%were reassembled into a dominant bacterial community as measured by 16S rDNA.[Results]The growth of A.annua seedlings inoculated with bacterial communities and pathogenic fungi or dominant bacterial communities and pathogenic fungi was significantly better than that of A.annua seedlings inoculated with pathogenic fungi during in vitro confrontation,which was evident in both enzymatic and non-enzymatic antioxidant assays.[Conclusions]The results suggest that the dominant bacterial community has a crucial role as a representative core microbial community of synthetic bacterial community,which can protect plants by interfering with the growth of phytopathogenic fungi mediated by chemical signals,and can be used as the main synthetic community of biocides to achieve the effect of biocontrol.

In Vitro Antifungal Activity of the Extract and Compound from Acorus tatarinowii Against Seven Plant Pathogenic Fungi

Acorus tatarinowii Schott is a traditional Chinese medicine plant and has multiple bioactivities in medicine and pesticide field. In this study, the antifungal compound 1,2-dimethoxy-4（2-propenyl） benzene was isolated from A. tatarinowii Schott by activity-directed isolation method, and the inhibitory activity of the extract and 1,2-dimethoxy-4（2-propenyl） benzene against seven plant pathogenic fungi was evaluated. The results showed that the extract and 1,2-dimethoxy-4（2- propenyl） benzene had high inhibitory activity against hyphal growth of Thielaviopsis paradoxa （de Seynes） V. Hohnel, Pestalotia mangiferae P. Henn., Fusarium oxysporum f. sp. niveum （E. F. Smith） Syn. et Hans., Alternaria alternate Tanaka, Colletotrichum musae （Berk et Curt） V. Arx, Sphaceloma fawcettii Jenk., and Mycosphaerella sentina （Fr.） Schroter. The EC50 values of extract were 1.6162, 1.6811, 1.1253, 3.5771, 1.7024, 2.2284, and 2.2221 g L^-1, respectively, and the EC50 values of 1,2-dimethoxy-4（2-propenyl） benzene were 0.1021, 0.0997, 0.0805, 0.1742, 0.1503, 0.1853, and 0.1924 g L^-1, respectively. 1,2-Dimethoxy-4（2-propenyl） benzene also inhibited spores germination of T. paradoxa （de Seynes） V. Hohnel and F. oxysporum f. sp. niveum （E. F. Smith） Syn. et Hans., with the inhibitory rates of 98.81 and 100% at a concentration of 0.4 g L^-1 after 8 h, respectively. 1,2-Dimethoxy-4（2-propenyl） benzene is a potential botanical antifungal agent for controling of plant fungal diseases.

Toxicity of Lanthanum to Pathogenic Fungi and Its Morphological Characteristics

The inhibitory effects of La on the mycelial growth of several soil-borne pathogenic fungi including Rhizoctonia solani , Pythium sp., Fusarium solani , Selerotinia sclerotiorum , and Fusarium oxyspoxum were studied in vitro. The results show that the mycelial growth was inhibited strongly by the La, with EC50 of 130 - 320 mg·L^-1 and EC95 of about 550- 40007 mg·L^-1 respectively. In addition, the morphological toxicity of La was studied by using the scanning electronic microscope. Treated by La, the mycelial growth of pathogenic fungi was significantly restrained, and the morphological characteristics were found to be abnormal, such as increased ramification, malformation, partial swelling and shrinking, and irregularly entangled mycelial block or rhizomorph.

A Preliminary Investigation of Pathogenic Fungi from Lotus(Nelumbo nucifera Gaertn)in Nanchang City

[Objectives]Lotus(Nelumbo nucifera Gaertn)is an economically important aquatic plant in China.Fungal disease is a serious problem in lotus cultivation.In this study,the pathogenic fungi on lotus in Nanchang City were investigated to lay the foundation for the disease control.[Methods]Lotus leaves and stems in ponds of Nanchang City were collected,the fungi on leave/stem spots were isolated and purified.Colonies morphological characters and ITS sequences were used to identify the strains.[Results]49 strains were isolated and identified to 20 species,belonging to 12 genera.[Discussion]15 species may firstly be reported on lotus in this study,i.e.,Alternaria angustiovoidea,Alternaria compacta,Alternaria ricini,Alternaria tenuissima,Arthrinium arundinis,Botryosphaeria dothidea,Curvularia spicifera,Diaporthe australiana,Diaporthe eres,Diaporthe tectonae,Epicoccum nigrum,Fusarium fujikuroi,Neofusicoccum parvum,Nigrospora sphaerica,and Phomopsis eucommii.

Antifungal Activity and Toxicity of Two New types of Polyene Antibiotics against Four Species of Rice Pathogenic Fungi

The antifungal activity and toxicity of new type polyene macrolide antibiotics-Antifungalmycin 702 and Fungichromin against Rhizoctonia solani, Hel- minthosporium sigmoideum Car. , Magnaporthe grisea and Ustilaginoidea virens were studied by mycelial growth rate method and mycelial dry weight method in vitro. The results showed that both of Antifungalmycin 702 and Fungichromin had strong antifungal activity against four speices of rice pathogenic fungi. Treated with 50 μmL Antifungalmycin 702 , the inhibitory rates of Antifungalmycin 702 agmnst R. solani, H. sigmoideum Cav., M. grisea and U. virens were 100%, 100%,91.20% and 68.10%, and EC50 and ECho were 7.00, 10.30, 14.41, 26.71 and 13.20, 17.66, 27.67,128.28 p.g/mL, respectively. Treated with 13 pg/mL Fungichremln, the inhibitory rates of Fungichromin against four speices of rice pathogenic fungi were 78.20%, 100%, 100% and 79.17%, and ECs0 and ECg0 were 1.47, 1.91, 2.37, 0. 20 and 41. （15, 3.92, 4.34, 135.54 gg/mL, respectively. The antifungal activity of Fungichromin was stronger than Antifungalmycin 702.

Selective Degradation of Mitochondria by Mitophagy in Pathogenic Fungi

Selective mitochondrial autophagy or mitophagy is an evolutionarily conserved cellular process that selectively degrades superfluous, damaged, and dysfunctional mitochondria. This process is believed to be a mitochondrial quality control system crucial for intracellular homeostasis. Recently, researchers developed a range of methods to induce mitophagy and a variety of assays to monitor this process. With these new methods, the research on mitophagy has been developed rapidly. In particular, some key receptors and regulatory factors in fungi have been identified, which provides a basis for further understanding of the mechanism of this process. Although it has been studied extensively in the model yeast Saccharomyces cerevisiae, mitophagy in pathogenic fungi remains poorly understood. However recent studies have shown that mitophagy is involved in the regulation of pathogenicity of pathogenic fungi, which greatly increases the importance of mitophagy. Therefore, it is necessary to review the current research on mitophagy in order to provide an accurate understanding of mitophagy and promote mitophagy research in the pathogenic fungi.

Effectiveness of Ringworm Cassia and Turmeric Plant Extracts on Growth Inhibition against Some Important Plant Pathogenic Fungi

Crude plant extracts of ringworm cassia, Cassia alata L. and turmeric, Curcuma longa L. were prepared by either hot water or organic solvents such as ethanol and ether. Various concentrations of the crude extract were then subjected to an in vitro test for their effectiveness on mycelia growth inhibition against some important plant pathogenic fungi such as Alternaria alternata, Colletotrichum gloeosporioides, Fusarium oxysporum fsp. lycopersici, Sclerotium rolfsii, Phytophthora infestans and Pythium sp. in comparison to commercial fungicides such as copper oxychloride and mancozeb. Reduction of the fungal growth was significantly obtained with C. longa extracts and the best median effective inhibitory concentration (IC50) value of 6.07, 6.50 and 7.13 mg/ml was from the ethanol extract for S. rolfsii, C. gloeosporioides and F. oxysporum fsp. lycopersici respectively. While all extracts from C. alata were almost the least effective against these fungi. The efficacy of C. longa extracts therefore, provided an alternative regime for the control of the fungal diseases and a promising appreciable choice for a replacement of chemical fungicides.

Phylogenetic Identification of Pathogenic Fungi from Apple in Batu City, Malang, Indonesia

Pathogenic fungi cause dying off of apple tree and giving negative impact to farmers. The objective of this study was to identify tree pathogenic fungi isolates from apple tree in the Batu City and to know phylogenetic relationship of the isolates based on the DNA sequences. The method in this study included subculture of three pathogenic fungi isolates (M1, M4, and MB1) using V8 Juice Agar medium for one to three days, continued with DNA isolation, amplification using ITS5/ITS4 primer, purification, sequencing, and sequence homology analysis of DNA amplicons of each isolates with reference isolates. The results showed similarity value of DNA sequence of the three isolates M1, M4, and MB1 of more than 99%. The three isolates M1, M4, and MB1 have a similarity value of DNA sequence with species of Pythium splendens of 99.84%, 99.67% and 99.83%, respectively. On the other hand similarity value between those isolates with Phytophthora was less than 76%. It was concluded that the three isolates are Py. splendens.

Isolation and Identification of Pathogenic Fungi in Kiwifruit during the Storage Period

[Objectives]The paper was to identify the pathogenic species of postharvest diseases of kiwifruit.[Methods]The pathogenic fungi were isolated from diseased kiwifruits during the storage period,and cultured to observe their culture traits and morphological characteristics.Molecular biological identification and pathogenicity test were carried out to confirm the main pathogens.[Results]There were 7 species of pathogenic fungi causing kiwifruit diseases during the storage period,including Alternaria spp.,Botryosphaeria dothidea,Phomopsis/Diaporthe spp.,Pestalotiopsis sp.,Pseudocercospora sp.,Colletotrichum sp.,and Botrytis cinerea.[Conclusions]The research will provide a scientific basis for the prevention and control of postharvest diseases of kiwifruit.

Isolation and Identification of Pathogenic Fungi Causing Decayin Luotian Chestnut during Late Storage Period and Research on Pathogenicity

In order to explore moldy mechanism of chestnut from Luotian County in storage process,the strains of pathogenic fungi were isolated from chestnuts after storage at room temperature for 70d.Six genera of fungi were found in chestnut through experimental identification,which were Ozoniumsp.,Fusarium sp.,Aspergillus sp.,Penicilliumsp.,Rhiopus sp.and Stachybotrys sp.,respectively.The re-inoculation tests had been conducted on pathogenic fungi whose isolating rate was greater than 10%.The result showed that the rest genera of fungi generally had no pathogenicity except Penicilliumsp.could infect non-injured chestnut with a lower moldy rate and lighter symptoms;but the moldy rate of strains was above 60% in injured inoculation and they showed heavy symptoms,among which the moldy rate of Ozoniumsp.and Aspergillus sp.were higher than 80%.The experimental results showed that injured chestnut were more likely to decay.Ozoniumsp.and Aspergillus sp.were important pathogenic fungi causing decay during storage process of chestnut.

Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic Fungi Causing External Ear and Skin Infections

Fifteen pathogenic fungi were isolated from patients with external ear infection like Aspergillus niger and 100 pathogenic fungi were isolated from patients with skin infections like Trichophyton mentagrophytes which was isolated from body skin infections （also four different species of the genus Trichophyton was isolated） as well as another fungus which is Microsporum audouinii was isolated from the head scalp, and Aspergillus ustus which was isolated as normal flora from infected skin inflammation These fungi are opportunistic that can cause skin infections and inflammation of the external ear which increases the severity and the length of the disease, so the effect of different concentrations of alcoholic extract of sage leaves was tested against these fungi and it was found that the extract had an inhibition effect on growth of the fungus A. ustus by 80.7% at a concentration of 20 mg/mL, while inhibited the fungus A. niger by 77.3% at the same concentration, whereas inhibited the fungus T. mentagrophyte by 53.3% at the same concentration, and the fungus Microsporum audouinii was inhibited at the same concentration by 86.6%, also the extract showed different inhibition values against the other four species of the fungus Trichophyton. The plant sage is considered as good antifungal towards studied fungi of otitis extema and skin diseases and can be used prospectively as antifungal antibiotics against fungi causing external ear infections and skin infections.

Identification of Plant-Pathogenic Fungi Using Fourier Transform Infrared Spectroscopy Combined with Chemometric Analyses

Identification of plant-pathogenic fungi is time-consuming due to cultivation and microscopic examination and can be influenced by the interpretation of the micro-morphological characters observed.The present investigation aimed to create a simple but sophisticated method for the identification of plant-pathogenic fungi by Fourier transform infrared(FTIR)spectroscopy.In this study,FTIR-attenuated total reflectance(ATR)spectroscopy was used in combination with chemometric analysis for identification of important pathogenic fungi of horticultural plants.Mixtures of mycelia and spores from 27fungal strains belonging to nine different families were collected from liquid PD or solid PDA media cultures and subjected to FTIR-ATR spectroscopy measurements.The FTIR-ATR spectra ranging from 4 000to 400cm-1 were obtained.To classify the FTIRATR spectra,cluster analysis was compared with canonical vitiate analysis(CVA)in the spectral regions of3 050~2 800and 1 800~900cm-1.Results showed that the identification accuracies achieved 97.53%and99.18%for the cluster analysis and CVA analysis,respectively,demonstrating the high potential of this technique for fungal strain identification.

Effect of Lanthanum on Mycelium Growth and Some Pathogenic Factors

Three soil-transmitted pathogenic fungi including Rhizoctonia solani, Fusarium solani, and Pythium sp. were selected to investigate the effect of lanthanum on their growth and the pathogenic enzymes using liquid culture. Variance analysis shows significant differences among treatments with different concentrations of lanthanum （Rhizoctonia solani F = 6.75 〉 F0.01= 5.99; Fusarium solani F = 18.1 〉 F0.01 = 5.99, Pythium sp. F = 23.29 〉 F0.01 = 5.99）. The inhibitory effect of lanthanum on pathogenic fungi increased with an increase in La concentration. The activities of the three pathogenic enzymes per gram mycelium were promoted remarkably. However, the quantity or the activities of the total enzymes were inhibited because of the strong inhibition of mycelium growth by lanthanum. Meanwhile, the effect of lanthanum on toxins of pathogenic fungi were studied using the seed germination experiment. Toxins of pathogenic fungi are influenced by lanthanum and the virulence decreases significantly with the increase of lanthanum concentration.

Effectiveness of entomopathogenic fungal strains against poplar/willow weevil(Cryptorhynchus lapathi L.)larvae

The poplar and willow weevil,Crypto rhynchus lapathi L.,a major universally destructive wood-boring insect has become one of the important quarantine pests that is extremely destructive to forestry development and needs to be controlled.Entomopathogenic fungi(EPF) are considered safe and friendly for humans and the environment and play important roles in controlling insect pest populations.In this study,the screening of entomopathogenic fungi for control of C.lapathi is reported through the evaluation of virulence of four fungal Beauveria bassiana(CFCC81428,CFCC83116,CFCC83486,CFCC87297)strains,one B.brongniartii(CFCC83487) strain and one Metarhizium anisopliae(CFCC88953) strain.The virulence of the different strains was appraised by correct mortality rate,cumulative mortality rate,median lethal concentration(LC_(50)) and median lethal time(LT_(50)).B.bassiana strains CFCC81428 and CFCC83116 were the most virulent among the six strains with a mortality up to 100%,and the LT_(50) were 2.7 and 3.1 days.Five conidia concentrations of three strains(CFCC81428,CFCC83116 and CFCC87298) that caused high virulence was screened for dose-relationship.Their effect on controlling C.lapathi larvae were also determined under field condition by brushing conidia suspensions on C.lapathi larvae infested in a poplar trunk.The cumulative rate in the field was lower than those obtained from the laboratory,but the order of the virulence of different strains did not change.Mortality in all three strains occurred at their highest concentration(1.0 × 10^(8) conidia mL^(-1)).Under field conditions,the CFCC81428 strain was the most effective,causing mortalities of 80.3% and 75.2%in two plots in Beipiao and Lindian counties,respectively,followed by CFCC83116(69.1%,66.6%) and CFCC87298(60.7%,59.3%).Based on our results,the B.bassiana strain CFCC81428 has the potential as a biological insecticide to control C.lapathi larvae.

Fungi Associated with Sand and Plants from Marine Coastlines: Potential Relevance for Human Health

The fungal community associated with beach sand and plants located along marine coasts are an under-studied area of research despite its potential relevance to human health. In this study, we isolated and identified the cultivable mycobiota associated with sand and plants collected along the coast of Gran Canaria (Spain) using culture-dependent and -independent methods. Clinically relevant species belonging to Cryptococcus spp. and related genera such as Naganishia and Papilotrema were isolated and identified from shoreline plants. Moreover, Candida tropicalis was isolated from beach sand, and Aspergillus fumigatus and Aspergillus terreus strains were associated with both types of samples (i.e., plants and beach sand). We conclude that beach sand and shoreline plants are potential reservoirs of fungi of high clinical interest. We recommend including beach sand and plants from the environment when assessing the quality of marine coastal systems. Our results open a framework for studying the natural marine environment and its role in the epidemiology of infectious diseases in order to more accurately manage public health.

Cand2 inhibits CRL-mediated ubiquitination and suppresses autophagy to facilitate pathogenicity of phytopathogenic fungi

The ubiquitin–proteasome system and the autophagy system are the two primary mechanisms used by eukaryotes to maintain protein homeostasis,and both are closely related to the pathogenicity of the rice blast fungus.In this research,we identified MoCand2 as an inhibitor of ubiquitination in Magnaporthe oryzae.Through this role,MoCand2 participates in the regulation of autophagy and pathogenicity.Spe-cifically,we found that deletion of MoCand2 increased the ubiquitination level in M.oryzae,whereas overexpression of MoCand2 inhibited the accumulation of ubiquitinated proteins.Interaction analyses showed that MoCand2 is a subunit of Cullin-RING ligases(CRLs).It suppresses ubiquitination by blocking the assembly of CRLs and downregulating the expression of key CRL subunits.Further research indi-cated that MoCand2 regulates autophagy through ubiquitination.MoCand2 knockout led to over-ubiquitination and over-degradation of MoTor,and we confirmed that MoTor content was negatively correlated with autophagy level.In addition,MoCand2 knockout accelerated the K63 ubiquitination of MoAtg6 and strengthened the assembly and activity of the phosphatidylinositol-3-kinase class 3 complex,thus enhancing autophagy.Abnormal ubiquitination and autophagy in DMocand2 resulted in defects in growth,conidiation,stress resistance,and pathogenicity.Finally,sequence alignment and functional an-alyses in other phytopathogenic fungi confirmed the high conservation of fungal Cand2s.Our research thus reveals a novel mechanism by which ubiquitination regulates autophagy and pathogenicity in phyto-pathogenic fungi.

Analytical characterization and structure elucidation of metabolites from Aspergillus ochraceus MP2 fungi

Objective:To isolate and characterize the bioactive secondary metabolites from Aspergillus ochraceus(A.ochraceus)MP2 fungi.Methods:The anti bacterial activity of marine sponge derived fungi A.ochraceus MP2 was thoroughly investigated against antagonistic human pathogens.The optimum inhibitory concentration of the fungi in the elite solvent was also determined.The promising extracts that showed good antimicrobial activity were subjected to further analytical separation to get individual distinct metabolites and the eluants were further identified by GC MS instrumental analysis.The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer(US)region of 5.8s rRNA using specific ITS primer.The novelty of the strain was proved by homology search tools and elite sequences was submitted to GENBANK.Results:Three bioactive compounds were characterized to reveal their identity,chemical formula and structure.The first elutant was identified asα-Campholene aldehyde with chemical fonnula C_(10)H_(16)O and molecular weight 152 Da.The second elutant was identified as Lucenin-2 and chemical fonnula C_(27)H_(30)O_(16)and molecular weight 610Da.The third elutant was identified as 6-Ethyloct-3-yl-2-ethylhexyl ester with Chemical fonnula C_(26)H_(42)O_4 with molecular weight 418 Da.Conclusions:The isolated compounds showed significant antimicrobial activity against potential human pathogens.Microbial secondary metabolites represent a large source of compounds endowed with ingenious structures and potent biological activities.

Antimicrobial Activity of Wild Plant Seed Extracts against Human Bacterial and Plant Fungal Pathogens

Five wild plant species belonging to different families (Chenopodium album, Plantago major, Elytrigia elongata, Filipendula ulmaria and Nigella sativa) widely spread in Russian Federation and the former USSR were evaluated for their ability to inhibit growth of two important human food-borne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes strain EGD-e) and eight plant pathogens (Alternaria alternata, Alternaria tenuissima, Bipolaris sorokiniana, Stagonospora nodorum, Fusarium solani, Fusarium oxysporum, Fusarium culmorum and Phytophtora infestans). To isolate biologically active compounds from seeds, a step-wise procedure including extraction with hexane, ethyl acetate, ethanol, and 10% acetic acid followed by reversed-phase HPLC was developed. Using disc-diffusion assay, the highest activity against E. coli O157:H7 was observed with extracts from F. ulmaria (hexane and ethyl acetate extracts and the unbound RP-HPLC fraction) and P. major (ethyl acetate extract and the unbound RP-HPLC fraction);E. elongate (the unbound RP-HPLC fraction) was less active. The extracts from P. major and E. elongate (the unbound RP-HPLC fractions) were equally highly active against L. monocytogenes, while those of F. ulmaria (the unbound RP-HPLC fraction) and N. sativa (hexane and ethyl acetate extracts) were less active against this pathogen. The dynamics of L. monocytogenes EGD-е and E. coli O157:H7 growth in the presence of two most potent extracts (RP-HPLC-unbound fractions of P. major and E. elongate and the hexane extract of F. ulmaria) was studied.

Characteristics of cross transmission of gut fungal pathogens between wintering Hooded Cranes and sympatric Domestic Geese

Migratory birds travel long distance and link various pathogens.Due to habitat degradation,wintering waterfowls forage together with poultry,increasing the risk of pathogen transmission between hosts.We investigated the fungal communities between wintering Hooded Cranes and Domestic Geese by high-throughput sequencing,and inferred the potential gut pathogens for both hosts at different wintering stages.The fungal community compositions were significantly different between seasons and between the hosts.The negative correlation was found between fungal diversity and pathogenic diversity,with higher fungal diversity and less pathogenic diversity in early stage and less fungal diversity and higher pathogenic diversity in late stage for both hosts.The dissimilarity of pathogenic community compositions decreased from early to late stage,with the quantity of overlapping potential pathogenic OTUs increasing along wintering periods between the two hosts,demonstrating the existence of pathogen cross transmission between the two hosts.Furthermore,the transmission pathway of avian pathogens was mainly from Hooded Cranes to Domestic Geese while the transmission direction of human pathogens was primarily from Domestic Geese to wild cranes.Based on above results,we argued that pathogen cross transmission and underlying outbreak risk of disease should be closely monitored in migratory birds and domestic poultry.

Sanger Sequencing Implementation in Clinically Ill Patients for Bacterial and Fungal Pathogens Identification

The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.