Yr5-virulent races of Puccinia striiformis f.sp.tritici possess relative parasitic fitness higher than current main predominant races and potential risk

Wheat stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most destructive fungal diseases of wheat,and seriously threatens safe production of the crop worldwide.In China,new races historically appeared and rapidly developed to be predominant races and have resulted in ineffectiveness and replacement of wheat resistance cultivars as well as massive reduction in yield.In the present study,the relative parasitic fitness of the two newlyemerged Yr5-virulent races(TSA-6 and TSA-9)were compared with those of four currently predominant Chinese races(CYR31,CYR32,CYR33,and CYR34)based on evaluation on 10 Chinese wheat cultivars.As a result,there were significant differences in the relative parasitic fitness parameters among overall tested races based on multiple comparison(LSD)analysis(P<0.05).The principal component analysis(PCA)of overall parasitic fitness parameters indicated that the sporulation ability,infection and spore survivability,expansion capacity,and potential pathogenicity were the most important parasitic fitness attributes of the tested races.Based on the establishment of extracted three principal components and a comprehensive factor score mathematical models,evaluations of the parasitic fitness attributes of tested races showed that the level of relative parasitic fitness of the tested six races was:CYR32(1.15)>TSA-9(0.95)>TSA-6(0.92)>CYR34(0.29)>CYR31(–1.54)>CYR33(–1.77).The results indicated that two Yr5-virulent races TSA-9 and TSA-6 possessed relative parasitic fitness higher than races CYR34,CYR31,and CYR33,but lower than race CYR32,and have potential risks in developing to be predominant races.Therefore,continual monitoring of both Yr5-virulent races,and their variants is needed.The use of wheat cultivars(lines)with Yr5 resistance gene singly in wheat breeding is essential for being avoided,and is suggested to combine with other effective stripe rust resistance genes.

Effect of Botryosphaeria berengeriana f.sp.piricola and Biocontrol Bacteria on the System of Antioxidant Enzymes in Pears

[Objective] The aim was to understand defense mechanism of pear after inoculated Botryosphaeria berengeriana f. sp. piricolan and mechanism of antioxidant enzymes of biocontrol bacteria. [Method] Pears were treated by Botryosphaeria berengeriana f. sp. piricolan and bio control bacteria, and the change of antioxidant enzymes were determined. [Result] The biocontrol bacteria had little effect on MDA;the content of MDA treated by B. berengeriana reached high peak in 48 h, was 10.22nmol/g which was 1.86 times of CK; the content of MDA treated by B. berengeriana and biocontrol bacteria reached high peak in 24 h, was 8.92 nmol/g which was1.62 times of CK. The content of SOD treated by biocontrol bacteria reached high peak in 48 h, was 126.69 U/[g（FW）·min] which was 1.54 times of CK; the contents of SOD treated by B. berengeriana as well as B. berengeriana and biocontrol bacteria reached high peak in 24 h, were 122.10 and 135.32 U/[g（FW）·min] which were 1.48 and 1.65 times of CK respectively; the contents of POD on biocontrol bacteria treatment, B. ana treatment as well as B. berengeriana and biocontrol bacteria treatment reached high peak in 24 h, were 385.34, 342.50 and 290.00 U/[g（FW）·min] which were 1.83, 1.62 and 1.38 times of CK respectively. The contents of CAT on biocontrol bacteria treatment, B. rengeriana treatment as well as B. berengeriana and biocontrol bacteria treatment reached high peak in 6 h, were 133.33,114.17 and 113.35 U/[g（FW）·min] which were 1.33, 1.14 and 1.13 times of CK respectively. The biocontrol bacteria had little difference in CK; the content of PPO of B. berengeriana treatment reached high peak in 12 h, was 81.86 U/[g（FW）·min]which was 1.76 times of CK; B. berengeriana and biocontrol bacteria treatment reached high peak in 24 h, was 70.00 U/[g（FW）·min] which was 1.50 times of CK.[Conclusion] B. berengeriana and biocontrol bacteria had more effect on MDA; both B. berengeriana and biocontrol bacteria could increase the excitation of SOD enzyme activity; both B. berengeriana and biocontrol bacteria could increase the excitation of POD enzyme activity; both B. berengeriana and biocontrol bacteria could increase the excitation of CAT enzyme activity; using biocontrol bacteria alone had not obvious effect on PPO, B. berengeriana could increase the excitation of PPO enzyme activity.

Virulence and Diversity of Blumeria graminis f.sp.tritici Populations in China

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0%were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0%to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated signiifcantly with geographical distance （R2=0.494, P 0.001）. In addition, isolates from Xinjiang appear to form a separate group. Signiifcant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.

Effects of Medium Nutrients on Fusarium oxysporum Schl. f.sp

[Objective] To study the effects of different culture conditions on the Fusarium oxysporurn SchL f. sp. [Method] Based on species identification of the pathogenic organism of Fusarium oxysporum Schl. f. sp, effects of different cultures and different nutrients on the mycelial growth and conidial production of Fusarium oxysporum SchL f. sp were studied. [Result] The mycelial growth and conidial pro- duction of Fusarium oxysporum SchL f. sp was different under different culture con- ditions. PDA medium was the most suitable medium for the mycelial growth and had the highest conidial production; and the mycelial grew the fastest on the medium with maltose as carbon source or peptone as nitrogen source, which also had the highest conidial production. [Conclusion] This study provided experimental basis for the study of Fusarium oxysporum SchL f. sp and also provided theoretical basis for the study and control of Fusarium oxysporum Schl. f. sp.

Identification of eight Berberis species from the Yunnan-Guizhou plateau as aecial hosts for Puccinia striiformis f.sp.tritici,the wheat stripe rust pathogen

Puccinia striiformis Westend.f.sp.tritici Erikss.(Pst)infects wheat and causes stripe rust.The rust is heteroecious with wheat as the primary uredinial and telial host and barberry(Berberis spp.)as the alternate pycnial and aecial host.More than 40 Berberis species have been identified as alternate hosts for Pst,and most of these are Chinese Berberis species.However,little is known about Berberis species or their geographic distributions in the Yunnan-Guizhou plateau in southwestern China.The Yunnan-Guizhou plateau is considered to be an important and relatively independent region for the evolution of the wheat stripe rust pathogen in China because the entire disease cycle can be completed within the region.In this study,we conducted a survey of barberry plants in the Yunnan-Guizhou plateau and identified the eight Pst-susceptible Berberis species under controlled conditions,including B.julianae,B.tsienii,B.veitchii,B.wilsonae,B.wilsonae var.guhtzunica,B.franchetiana,B.lepidifolia and B.pruinosa.These species are reported here for the first time to serve as alternate hosts for the wheat stripe rust pathogen under controlled conditions.

Effects of Spores and Crude Toxins of Helminthosporium gramineum Rabenh f.sp.echinochloae on the Activity of Defensive Enzymes in Barnyardgrass

[Objective] This study aimed to explore the effects of spores and crude toxins of Helminthosporium gramineum Rabenh f. sp. echinochloae（HGE） on the ac- tivity of defensive enzymes of barnyardgrass [Echinochloa crus-galli （L.） Beauv.]. [Method] The effects of spores and crude toxins of HGE, as well as the mixture of spores and crude toxins on the activity of defensive enzymes in barnyardgrass were determined under laboratory conditions. [Result] Spores and crude toxins of HGE had varying degrees of effects on PAL and POD activity, and no obvious effect on SOD activity in barnyardgrass. In addition, spores and toxins had some similar im- pacts on the defensive enzymes in barnyardgrass. [Conclusion] Since toxins have similar effects on the hosts as spores of fungal pathogen do, they can be a substi- tute for the fungal pathogen in studying the partial pathogenic mechanism of this pathogen due to its complexity in pathogenic process.

Tolerance of New Introgressive Hybrid and Backcross Forms Pathogenic Micromitisms (<i>Verticillium dahliae</i>Kleb and Fusarium oxysporum f.sp. vasinfectum)

The article is based on the use of experimental polyploidy method, with the introduction of new introgressive hybrid forms combining several species genotype with pathogenic Verticillium dahliae Kleb. and the effects of mycotoxins separated from the Fusarium oxysporum f.sp. vasinfectum micromicette on the yield of plant seeds. New artificial complex hypertension forms based on experimental polyploidy Verticillium dahliae Kleb. and Fusarium oxysporum f.sp. vasinfectum combine the potential of resistance to mycotoxins separated from microcrystals, making a tremendous contribution to the selection of new varieties and to the effectiveness of selection as a result of the use of genetic selective research as genetic-selective genetic-selector studies.

Research Advances in New Race Ug99 of Puccinia graminis f.sp. tritici

This paper reviewed the research advances of Ug99 and its resistance breeding from the aspects of its discovery, race variation, pathogenicity, distribution, spread, exploration of relative resistant genes, linked molecular marker selection and resistance breeding strategies, to provide basis for comprehensive understanding of the new Puccinia graminis f. sp. tritici race Ug99 and its potential threat to wheat production. Ug99 is a new Puccinia grarninis f. sp. tritici race with high variability, strong pathogenicity and rapid spread speed, which is likely to cause global damages to world wheat production. We should strengthen the exploration and utilization of new resistance genes in wheat and relative species and breeding of new wheat varieties with durable resistance, to control and prevent damages caused by Ug99 and its variants.

Development of SSR Markers for a Phytopathogenic Fungus,Blumeria graminis f.sp.tritici,Using a FIASCO Protocol

Simple sequence repeats （SSR） have been widely used as molecular markers due to their abundance and high polymorphism, However, up to now, the SSR markers had not been developed in the obligate biotrophic phytopathogenic fungus, Blumeria graminis f.sp. tritici. From （AC）10 and （AG）10 enriched genomic libraries for Bgt, 25 primer pairs were designed using the FIASCO （fast isolation by AFLP of sequences containing repeats） protocol. Five primer pairs exhibited polymorphism with allelic diversity from two to seven alleles and produced 29 alleles in a survey of 90 isolates collected from six provinces （cities） in China, while the others displayed monomorphic. Levels of observed heterozygosity ranged from 0.000-0.044 （mean 0.025） and expected heterozygosity ranged from 0.297-0.816 （mean 0.538）. These molecular markers provide a novel source to genetic diversity assays and to genetic and physical mapping ofBgt. SSR markers of Bgt need to be further explored.

Study on Control Effect of Invasive Banana Fusarium Disease(Fusarium oxysporum f.sp.cubense tropical race 4)by Different Varieties and Fertilization Treatments

[Objective]The paper was to understand the field resistance performance of different banana varieties and the prevention and control ef-fect of different fertilizers,so as to provide technical reference for the prevention and control of Fusarium wilt.[Method]Field trials were set up with three treatments:shrimp peptide organic fertilizer+shrimp peptide special protection+shrimp peptide fruit Yekang(simplified as shrimp peptide organic fertilizer treatment),conventional organic fertilizer+microbial preparations(simplified as microbial treatment),and conventional or-ganic fertilizer(simplified as control).Four different banana varieties of Brazilian banana,Guijiao No.1,Nantianhuang,and Yunjiao No.1 were se-lected for the field trial.The disease incidence of Fusarium wilt and the control effects of three fertilizers were investigated during four time periods.[Result]The disease incidence of four varieties in three treatments varied.The disease incidence of Nantianhuang and Yunjiao No.1 were signifi-cantly lower than that of other two varieties.There was also significant difference in disease incidence of three treatments.The disease incidence from high to low was control>shrimp peptide organic fertilizer treatment>microbial treatment.The average monthly TR4 pathogen content in heavily infected banana plantation was more than 2000 copies,while the highest one reached 15148.9 copies.[Conclusion]Microbial agents reduced the disease incidence of Fusarium wilt to some content.Nantianhuang and Yunjiao No.1 showed the highest disease resistance compared with other varieties.However,their resistance needs to be further improved before practical application.

Efficacy of Some Fungal Seed Dressers in Controlling Fusarium oxysporum f.sp.Cicer on Chickpea under Artificial Infection Conditions

Seeds of chickpea cv.Ghab3 were treated with three fungicides;Oxycure,Topsin and Vitaflo,and infected by Fusarium Oxysporum f.sp.Ciceri at culturing under a glasshouse condition.The results showed that Oxycure(Oxychloride)caused a significant increase in seed germination compared with other treatments,but it could not provide this protection at the post-germination phase,resulting in a significant increase in seedling mortality to 42.1%at 15 days after germination,compared with the other fungicides.In addition,Topspin(Thiophanate-methyl)was an effective protectant at germination stage,and in the seedling one too,since the percent of seedling mortality did not exceed 11.11%,whereas,Vitaflo had the least effect in protecting seeds from fungal infection,but with a significant difference with the positive control,as the percentage of non-germinated seeds was 41.66%and 62.5%,respectively.

Random Amplified Polymorphic DNA （RAPD） Analysis on Blumeria graminis f.sp. tritici Isolates Collected in Central Gansu Province, China

Twenty isolated strains of Blumeria graminis f.sp. tritici collected from central Gansu province were studied with random amplified polymorphic DNA （RAPD） analysis. PCR amplifications using nine random primers generated a total of 81 bands, of which, 54 were polymorphic. The total percentage of polymorphic bands varied from 50.0% to 88.9%. The average percentage based on RAPD patterns was approximately 66.7%, which indicated high heredity differentiation among isolates. Clustering analysis showed that the polymorphism of the twenty isolates was related to geographical origins but had little relationship with the physiological race.

Pathotypes and Genetic Diversity of Blumeria graminis f.sp.hordei in the Winter Barley Regions in China

Barley powdery mildew caused by Blumeria graminis f. sp. hordei （Bgh） is one of the most destructive foliar diseases of barley in the winter barley region of China. The evaluation and assessment of the virulence and diversity of Bgh populations help to determine effective sources of resistance to the pathogen. 515 isolates were collected from seven populations of Bgh on cultivated barley in seven geographically distant locations in 2006. Their virulence was determined by inoculation onto 26 differential host lines. All of the isolates belonged to 58 pathotypes and 13 of which included 81% of these isolates. The most abundant pathotype was pathotype 0047 （18.3%）, the second most abundant was pathotype 0045 （11.8%） and the third most abundant was pathotype 0057 （7.8%）. Most of virulent genes investigated in this study showed similar frequencies in the seven different areas. These indicate that the seven locations may be in a uniform epidemiological region and barley cultivars in these areas may have similar genetic background. Diversities within these populations and distances between these populations measured by KOIND package were different. Correlations were not found between the genetic distance and the geographical distances between different locations. This suggested that long distance spread and local epidemics existed in the major winter barley growing regions in China.

A FIASCO-Based Approach for Detection and Diagnosis of Puccinia graminis f.sp.tritici in China

Stem or black rust of wheat, caused by the fungus Puccinia graminis Pers. f. sp. tritici Eriks.＆E. Henn. （Pgt）, has historically caused severe losses to wheat （Triticum aestivum L.） production worldwide. In the Fujian and Guangdong provinces of China, six moderate-to-severe epidemics of wheat stem rust have occurred, which caused destructive losses of wheat between 1949 and 1966, although these were brought under control by integrated management. A rapid and reliable detection of the pathogen will contribute to the accurate forecast and seasonal control of this disease. The objective of this study was to develop a diagnostic molecular marker generated from simple sequence repeats （SSR） for the early rapid identiifcation of P. graminis. The genomic DNA of P. graminis, Puccinia striiformis, Puccinia triticina and seven other species was ampliifed by a pair of SSR primers generated by the FIASCO （fast isolation by AFLP sequences containing repeats） enrichment protocol. The primer set Pgtw （f）/Pgtw （r） generated a polymorphic pattern displaying a 330-bp DNA fragment speciifc for P. graminis whereas no DNA fragment was obtained from other non-target wheat fungal pathogens. The detection limit of the primer was 1 ng DNA in a 25-mL PCR reaction. The SSR markers of P. graminis can also be used to detect the presence of latent hyphae in Pgt-infected wheat leaves as early as 30 h post-inoculation. A rapid approach to distinguish P. graminis from similar pathogenic fungi would be anticipated in further study.

Biochemical and Physiological Changes of Three Watermelon Cultivars Infested with Fusarium oxysporum f.sp.niveum

The dynamic changes in membrane permeability, activities of disease-related enzymes, and contents of pathogenesis-relevant chemical compounds and photosynthetic pigments in root cells of three watermelon cultivars were studied after inoculation with Fusarium oxysporum f. sp. niveum at seedling stage. The results showed that the capacity of self-regulating and returning to normal status of cultivar Kelunsheng (resistant) was greater than that of a susceptible cultivar, Zaohua, in terms of malonaldehyde (MDA) content, relative conductivity, and activity of superoxide dismutase (SOD) and catalase (CAT). The resistant cultivar maintained a higher activity of dehydrogenase, higher content of vitamin C(Vc) , and relatively lower content of soluble sugar than the susceptible cultivar. The content of soluble protein was higher in the resistant cultivar than that in the susceptible one at day 1 after inoculation. The capacities of the resistant cultivar to inhibit chlorophyll deterioration and maintain a higher carotenoid content were significantly stronger than those of the susceptible cultivar.

Genetic analysis of the maximum germination distance of Striga under Fusarium oxysporum f.sp.strigae biocontrol in sorghum

Maximum germination distance（MGD） is an important component of Striga resistance in sorghum. The objective of this study was to determine gene action influencing MGD of Striga hermonthica and Striga asiatica among selected sorghum lines treated with a biocontrol agent, Fusarium oxysporum f. sp. strigae（FOS） for effective breeding with Striga resistance, and FOS compatibility. Twelve sorghum genotypes were selected based on their Striga resistance, FOS compatibility, and superior agronomic performance. Selected genotypes were crossed using a bi-parental mating design to generate six families for genetic analysis. Agar-gel assays were used to determine low haustorium initiation factor（LHF） using the 12 parental lines, their F_1 progenies, backcross derivatives, and F_2 segregants in two sets. One set had S. hermonthica seed and the other one had S. asiatica seed. Both were treated with and without FOS. Genotypes were evaluated using a split-plot design with three replications and MGD data were recorded followed by generation mean analysis. FOS reduced MGD by 1cm under both S. hermonthica and S. asiatica infestations. Additive, dominance, and epistatic gene actions were involved in the control of MGD of the two Striga species in the evaluated populations. On average, the relative contribution of additive, additive×additive and dominance×dominance genetic effects on the MGD of S. hermonthica and S. asiatica, with FOS, were 20, 33, and 36%; and 21, 32, and 35%, respectively. Breeding methods exploiting these genetic effects may provide enhanced response to selection for Striga resistance and FOS compatibility in integrated Striga management（ISM） programmes.

Studies on the control of Fusarium oxysporum f.sp. cubense with Trichoderma

One hundred and fifty one isolates of Trichoderma were collected mainly from more than 40 soil samples and other materials in Guangdong Province (including Chigang, Zhanjiang, Wuchuan, Panyu, Zhaoqing, Dongguan, Humen, Qingyuan, Guanzhou) and the soil samples were also from different plant rhizosphere (including rice, different fruits and different vegetables). It was shown that 39 isolates of Trichoderma grew faster than other isolates using growth velocity experiments. The 39 isolates could effectively inhibit Fusarium oxysporum f.sp. cubense (E.F.Sm) Sny.&Hans. by dual cultural experiments. The inhibited activity included the antifungal activities of its metabolite, mycoparasitic activities and the lytic enzymes by dual culture, electronic microcopy and enzyme assay. At present, studies on the taxonomy of the 151 isolates of Trichoderma are carried out in our experiments, some Trichoderma species aggregates will be identified according to the taxonomy system revised by Rifai and Bissett.

Study Genetic Variation Using DNA Molecular Markers and Identification Physiological Races of Wheat Stripe （yellow） Rust Puccinia striiformis f.sp tritici during 2010-2014 in Some Regions of Syria

Yellow Rust （stripe） rust （Puccinia striiformis West. f. sp. tritici） is one of the most epidemic diseases infect wheat in cold and wet regions. In 1988, this disease caused a loss of seasonal production amounted 70% on wheat variety Mexipak in Syria, and recurrent infection in 2010, caused by a virulent race called Yr27, caused a considerable loss in the production of bread wheat cultivars （Cham 8, Cham 6 particularly） amounted 90%. Recently, 15 races of yellow rust had been addressed in Syria for seasons 2010-2014; 159E256, 166E254, 166E256, 255 E112, 0 E0, 64 E 6, 230 El50, 0 E 18, 198 El30, 166 El50, 102 El60, 128 E0, 126 El50, 214E150, and 6E16. The race 6E16 was the most frequent during the two seasons, while the race 255El12 was the most virulent, followed by the race 230E222 and the race 0E0 was the weakest one. This study revealed the presence of fourteen newly observed races in Syria. Molecular Variance Analysis of Molecular Variance （AMOVA） of 55 yellow rust Puccinia striiformis f.sp tritici isolates examined by Amplify Fragment Length Polymorphism （AFLP） revealed high genetic variation within population, and the dimensional scale analysis （MSD） and tree diagram showed that the Syrian yellow rust isolates were clustered in three groups： the first group contained isolates derived from durum wheat, the second one contained bread wheat isolates, but the third was made of isolates derived from both durum and bread wheat species.

High-throughput RNA sequencing reveals differences between the transcriptomes of the five spore forms of Puccinia striiformis f.sp.tritici,the wheat stripe rust pathogen

The devastating wheat stripe(yellow)rust pathogen,Puccinia striiformis f.sp.tritici(Pst),is a macrocyclic and heteroe-cious fungus.Pst produces urediniospores and teliospores on its primary host,wheat,and pycniospores and aeciospores are produced on its alternate hosts,barberry(Berberis spp.)or mahonia(Mahonia spp.).Basidiospores are developed from teliospores and infect alternate hosts.These five spore forms play distinct roles in Pst infection,disease development,and fungal survival,etc.However,the specific genes and mechanisms underlying these functional differences are largely unknown.In this study,we performed,for the first time in rust fungi,the deep RNA sequencing to examine the transcriptomic shift among all five Pst spore forms.Among a total of 29,591 identified transcripts,951 were specifically expressed in basidiospores,whereas 920,761,266,and 110 were specific for teliospores,pycniospores,aeciospores,and urediniospores,respectively.Additionally,transcriptomes of sexual spores,namely pycniospores and basidiospores,showed significant differences from those of asexual spores(urediniospores,teliospores,and aeciospores),and transcriptomes of urediniospores and aeciospores were more similar to each other than to the three other spore forms.Especially,the basidiospores and pycniospores which infected the berberis shows wide differences in the cell wall degrading-enzymes and mating and pheromone response genes.Besides,we also found that there are 6234 differential expressed genes between the urediniospores and pycniospores,while only have 3 genes have alternative splicing enents,suggesting that differential genes expression may make more contribution than AS.This comprehensive transcriptome profiling can substantially improve our understanding of the developmental biology of the wheat stripe rust fungus.

Development of a Colorimetric Loop-mediated Isothermal Amplification Assay for the Visual Detection of Fusarium oxysporum f.sp. melonis

Fusarium wilt of melon, Fusarium oxysporum f.sp. melonis is one of the most important diseases, causing tremendous losses in melon growing areas in Iran. There is a real need for a rapid and inexpensive assay to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. One of the procedures designed for detection of this disease is loop-mediated isothermal amplification(LAMP)assay; its efficiency has been contrasted with polymerase chain reaction(PCR). The translation elongation factor 1-alpha gene is basically used for designing the LAMP(i.e. F3, B3, FIP, and BIP) together with PCR(F and B). Using hydroxynaphthol blue(HNB) dye, LAMP was placed in a water bath after the optimization was done. The results show LAMP is an advantageous method because it is highly sensitive(100-fold), quite cheap,user-friendly, and safe; in addition, it is performed quickly by visual detection and does not require DNA extraction(in direct-LAMP). The LAMP is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually.