Diversity of Fusarium Species Isolated from Weeds and Plant Debris in Croatia

Weeds are alternative hosts of plant pathogens and when colonized may not exhibit disease symptoms. In 2008 and 2009, samples of weeds and plant debris were collected from 12 locations in eastern Croatia, and 300 Fusarium isolates colonizing them were identified. Strains were grouped and identified based on morphology and amplified fragment length polymorphism (AFLP) patterns. Portions of the β‐tubulin and translocation elongation factor 1‐α genes were sequenced from representative strains of each group to confirm the identifications. Fourteen Fusarium species were identified with F. graminearum (20%), F. verticillioides (18%), F. oxysporum (16%), F. subglutinans (13%) and F. proliferatum (11%) all present as more than 10% of the population. Fusarium acuminatum, F. avenaceum, F. concolor, F. crookwellense (F. cerealis), F. equiseti, F. semitectum, F. solani, F. sporotrichioides and F. venenatum, were all present at frequencies < 8%. Our results indicate that economically important Fusarium spp. may be isolated from numerous alternative hosts during the off season and that weeds and plant debris can serve as a reservoir of genetically diverse inoculum.     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

我国东北地区玉米穗腐镰孢菌的种类及其分离频率

从辽宁、吉林、黑龙江三省采集的43份玉米病穗上共分离获得327株镰孢菌,根据形态特征鉴定属于7个种,其分离频率分别为半裸镰孢菌(Fusarium semitecturrt)37.77%,尖孢镰孢菌(F.oxysporum)3.51%,拟轮枝镰孢菌(F.verticillioides)3.57%,克鲁克威尔镰孢菌(F.c...     

Fusarium Species Associated with Mango Malformation in Peninsular Malaysia

Mango malformation has become the most important global disease on mango. Fusarium species previously associated with this disease include F. mangiferae, F. mexicanum, F. sterilihyphosum, F. proliferatum, F. subglutinans and F. tupiense. A few strains of F. proliferatum have been reported from Malaysia, but in this study, we report the results of more extensive sampling. The recovered strains were evaluated with morphology, mating tester strain cross‐fertility, amplified fragment length polymorphisms (AFLPs), and partial DNA sequences of the genes encoding translation elongation factor 1‐α (tef‐1α) and β‐tubulin (tub‐2). Amongst the 43 strains evaluated, three species were identified – F. proliferatum, F. mangiferae and F. subglutinans – with F. proliferatum being the most frequent (69%). None of the Fusarium species that appear to originate in the Americas were recovered in Malaysia, which suggests special measures may be warranted to keep these species from entering the country.     

中国的膨孢组镰孢菌(英文)

膨孢组镰孢菌Fusarium在自然界中广泛分布。该组包括4个种:木贼镰孢菌F.equiseti,藨草镰孢菌F.scirpi,长脚镰孢菌F.longipes和紧致镰孢菌F.compactum。这4个种均产生腹背不平行弯曲的大孢子。分离获得并描述了其中的3个种。木贼镰孢菌F.equiseti是镰孢菌中最常见的种之一,它产生典型的腹背不平行弯曲的大孢子,大孢子的顶细胞和足跟状基细胞明显伸长,菌落因缺乏红色素而呈黄褐色。藨草镰孢菌F.scirpi是比较少见的种类,它的典型特征是在典型的十字形产孢细胞上产生大量的小型分生孢子。由于它在PDA培养基上容易发生小孢子缺乏型的变异,因此,常常被错误地鉴定为木贼镰孢菌F.equiseti。长脚镰孢菌F.longipes的大孢子最容易与其他种的大孢子区分,它的顶细胞和基细胞均极度延长。当长脚镰孢菌F.longipes菌落因为变异而失去产生红色素的能力时,也容易与木贼镰孢菌F.equiseti混淆。基于该组大孢子的典型特征,作者将锐顶镰孢菌F.acuminatum排除在该组之外。     

Plant extracts abated pathogenic Fusarium species of millet seedlings

Millet is an important crop threatened by soil-borne pathogenic fungi worldwide, but the position of these soil-borne fungi most especially Fusarium has not been evaluated in south-western Nigeria alongside their biocontrol using plant extracts. This study investigates the effects (in vitro and in vivo) of Moringa oleifera, Manihot esculenta (peels) and Senna alata at 5, 10 and 15% g/ml concentrations on Fusarium anthophilum, Fusarium verticillioides, Fusarium oxysporum and Fusarium scirpi. The in vivo experimental layout was arranged in a completely randomised design and all obtained data, both in vitro and in vivo, were statistically analysed using Minitab version 15. The extract of Moringa oleifera, Manihot esculenta (peels) and S. alata at 5, 10 and 15% g/ml concentrations showed significant (p?<?0.05) antagonistic effect in vitro and in vivo on F. anthophilum, F. verticillioides and F. oxysporum. Moringa oleifera and S. alata at 5 and 10% g/ml significantly (p?<?0.05) reduced the disease incidence and disease severity of F. scirpi on millet seedlings. Thus, Moringa oleifera appears to be the most effective extract followed by Manihot esculenta, while S. alata was the least effective plant extract. Hence, the effectiveness of plant extracts on pathogenic Fusarium of millet justified their management strategy geared towards sustainable millet growth in Nigeria.     

Occurrence of Toxic Hexadepsipeptides in Preharvest Maize Ear Rot Infected by Fusarium poae in Poland

Twenty‐seven preharvest maize ears affected by Fusarium poae rot (disease score 36–100%) were selected in 1998 and 1999 in Poland and examined for the occurrence of toxic hexadepsipeptides: beauvericin (BEA), enniatin A, enniatin B and enniatin B₁. The identification of F. poae was confirmed by sequence analysis of variable internal transcribed spacer regions and compared with NCBI gene bank DNA sequences. Chemical analyses were performed by HPLC‐MS. In 27 ears infected by F. poae were detected: BEA (trace to 46 μg/g) in 18 samples, enniatin A (trace to 37 μg/g) in nine samples, enniatin B (trace to 47 μg/g) in 15 samples and enniatin B₁ (trace to 25 μg/g) in 12 samples. When 20 strains of F. poae isolated from these samples were cultured on rice, all produced BEA (1.9–75 μg/g), three enniatin A (1.8–2 μg/g), 12 enniatin B (1.1–5.1 μg/g) and eight enniatin B₁ (1.2–5.2 μg/g). Occurrence and quantification of enniatin A, enniatin B and enniatin B₁ and their co‐occurrence with BEA in maize kernels is reported for the first time.     

Nivalenol and 15‐acetyldeoxynivalenol Chemotypes of Fusarium graminearum Clade Species are Prevalent on Maize throughout China

Fusarium graminearum clade species are among the main causative agents of Gibberella ear rot (GER) in maize and responsible for the various trichothecene mycotoxins accumulated in contaminated maize grains. In this study, a total of 620 isolates from diseased maize ears collected from 59 districts in 19 provinces throughout China, previously identified morphologically as Fusarium graminearum clade, was genetically characterized at the species level based on SCAR (Sequence Characterized Amplified Region) and for their potential capability of mycotoxin production using the genetic chemotyping assay. The results showed that 359 isolates were F. asiaticum (SCAR 5), which consisted of 97% nivalenol (NIV)‐chemotypes, 0.8% 3‐acetyldeoxynivalenol (3‐ADON)‐producing isolates and 2.2% 15‐acetyldeoxynivalenol (15‐ADON) producers, whereas the remaining 261 isolates were identified as F. graminearum sensu stricto (SCAR 1), all of which produced 15‐ADON mycotoxins. This high proportion of NIV producers present in F. asiaticum is different from the chemotype patterns in F. asiaticum populations isolated from wheat and barley, where DON and its acetylated chemotypes were the predominant mycotoxins. Moreover, the majority of NIV producers (59.1%) and all the 3‐ADON‐producing strains were derived from the warmer regions in southern China, whereas most of the 15‐ADON‐producing strains (78.4%) were isolated from the colder regions in northern China. Our study is the first report of NIV chemotypes of F. asiaticum and 15‐ADON chemotypes of F. graminearum sensu stricto that were associated with the GER of maize in China.     

库姆塔格沙漠不同区域种子植物物种组成与区系特征研究

库姆塔格沙漠是我国西北荒漠地区较典型的沙漠之一,本文基于实地调查并结合遥感资料,对该地区植被的物种组成及种子植物区系特征进行了研究。结果表明:(1)库姆塔格沙漠地区共有种子植物29科91属143种;该地区植物物种贫乏、生活型简单,主要以灌木、一年生及多年生草本植物为主。(2)库姆塔格沙漠地区的植物组成受水分、高温以及土壤盐分等因子影响,形成了3个主要的种子植物分布区(阿奇克谷地、阿尔金山山前洪积扇戈壁、西湖湿地),且各区的物种组成差异显著。(3)从3个主要植物分布区的物种组成与生活型组成来看,阿尔金山山前洪积扇戈壁区域植物生活型比阿奇克谷地和西湖湿地丰富;因阿尔金山山前洪积扇戈壁环境复杂,在3个区域中其物种相对丰富,科、属、种的种类较多;阿奇克谷地和西湖湿地植物的属、种相似度较高;3个区域的植物属、种总体上相似度较低。(4)库姆塔格沙漠地区种子植物区系表征科主要为藜科(Chenopodiaceae)、菊科(Compositae)、麻黄科(Ephedraceae)等;3个主要植物分布区科的分布型均以世界分布为主,属的分布型以北温带分布和地中海区、西亚至中亚分布为主,具体表现为:阿奇克谷地和西湖湿地植物属的分布型以地中海区、西亚至中亚分布为主,阿尔金山山前洪积扇戈壁植物属的分布型以北温带分布为主。(5)库姆塔格沙漠地区种子植物以旱生种所占比例较高,物种类型具有明显的残遗性;由于环境条件特殊,形成了库姆塔格沙漠地区特有的植物类群和区系特征。本研究结果为库姆塔格沙漠地区植物组成及区系研究提供了基础资料。     

Investigation and a New Evaluation Method of the Resistance of Maize Hybrids grown in Hungary to Fusarium moulds

Thirty maize hybrids grown in Hungary representing groups FAO 200–299, FAO 400–499 and FAO 500-were studied in order to obtain information about genotypic resistance to Fusarium moulds. The plants were grown on an experimental farm and were inoculated using the toothpick method with Fusarium graminearum and Fusarium culmorum . In addition maize grain meals were also inoculated with isolates of moulds. Measurements were made of the mould-covered surface area of the ears 9 weeks after inoculation and of the zearalenone and T2-toxin content of the inoculated maize meals. Large differences among hybrids were observed for the mould-covered area of the ear surface (2.00–38.88%), the zearalenone content (4.20–71.20 mg/kg) and the T2-toxin content (1.60–122.50 mg/kg). Relatively poor correlation ( r =0.489) was found between the area of mould covering the ear surface and mycotoxin content of maize. Bearing in mind that the user of feed grain is interested in obtaining a feed with the lowest mycotoxin content, a new method of evaluation of hybrids which uses a toxin–mould index (TMI) was introduced. This index is calculated on the basis of both growth rate of moulds and their toxin-producing activity. Although a decreasing tendency in resistance of hybrids with a longer growing vegetation period could be observed, resistant genotypes were found in every FAO group, confirming the views that in addition to the influence of duration of the vegetation period on the resistance, genetic factors may also play a significant role.     

Studies on the Characteristic of Element Contents in the Dominant Plant Species of the Three gorges Region in China

The characteristics of the contents of 20 elements (Al, Fe, Mn, Ca, Mg, K, S, Si, P, Cd, Cu, Zn, Ti, Ni, Sr, Mo, Na, B, Cr, V) in 16 plant species collected from the Three Gorges Region in China were investigated. The average contents of Ca, K and Mg were higher than 1 000 μg·g-1, that of Al, P, Si, Fe, S and Mn ranged between 100—1 000 μg·g-1 and Ti, Cu, Ni, Cr, Mo, Cd and V were less than 10 μg·g- 1. The level of Na content was less than that of the reported. The main character of the element contents was of the Ca> K type. The contents of P, S, Ca and K in different plant samples showed a normal distribution pattern, while Al and Mn showed a elements lognormal distribution pattern. Plant species differed greatly in the element contents. On analyzing the coefficient of variation (C. V., % ), Al, Mn, Mg, Ni, Sr and Fe had higher C.V., while the C.V. of K, S, P, Cr, Cd and Cu was less than 60%, and Cu had the lowest C.V. The correlations between Al and Fe, Al and Ti, Al and Cr, A1 and V, Cd and Sr, Cd and Mo, Fe and V, Zn and Cr, Ni and Sr, Mg and Ni, Mo and Sr, Ca and Sr, Cr and Mo, Na and Mg, Na and P, P and S were statistically significant in different plant species. The classification of the 16 plant species and 20 dements by two-way indicator species analysis (TWINSPAN) method may suggest the difference in dement contents of the different plant species.     

Differences in Earthworm Densities and Nitrogen Dynamics in Soils Under Exotic and Native Plant Species

Previous studies of the invasion of two exotic plants – Berberis thunbergii and Microstegium vimineum – in hardwood forests of New Jersey have shown a significant increase of pH in soils under the invasive plants as compared with soils from under native shrubs (Vaccinium spp). We present a further investigation of soil properties under the exotic plants in question. We measured the densities of earthworms in the soil under the two exotics and the native shrubs in three parks in New Jersey. In the same populations we also measured the extractable ammonium and nitrate in the top 5 cm of the soil, as well as the respiration of the soils and the potential rates of mineralization (aerobic lab incubation). In addition, we measured the nitrate reductase activity in leaves of the two exotic plants and several native shrubs and trees. Although there were differences between parks, we observed significantly higher earthworm densities in the soil under the exotic species. The worms were all European species. Soil pH, available nitrate and net potential nitrification were significantly higher in soils under the two exotic species. In contrast, total soil C and N and net ammonification were significantly higher under native vegetation. Nitrate reductase activities were much higher in the leaves of exotic plants than in the leaves of native shrubs and trees. Changes in soil properties, especially the change in nitrogen cycling, associated with the invasion of these two plant species may permit the invasion of other weedy or exotic species. Our results also suggest that even if the two exotic species were removed, the restoration of the native flora might be inhibited by the high nitrate concentrations in the soil.     

Fusarium Species Isolated from Common Weeds in Eggplant Fields and Symptomless Hosts of Fusarium oxysporum f. sp. melongenae in Turkey

Thirteen species of weed plants were collected between May and September in 2010 and 2011 from eggplant fields representing 11 distinct locations covering a wide geographical area of Turkey. Weeds are potential hosts of many plant pathogens and may not exhibit disease symptoms when colonized. Fusarium spp. were isolated from five monocotyledonous species and eight dicotyledonous species. A total of 212 isolates recovered from weeds were assigned to eight Fusarium species on the basis of morphological characteristics. F. oxysporum was the most frequently isolated species (29.7%), followed by F. solani (19.8%), F. graminearum (13.7%), F. verticillioides (12.7%), F.equiseti (9.9%), F. avenacearum (8.0%), F. proliferatum (3.8%) and F. subglutinans (2.4%). The F. oxysporum isolates from different weed hosts were characterized by means of pathogenicity and vegetative compatibility grouping (VCG) tests. Among these, 29 isolates were found to be pathogenic to eggplant cv. Kemer and re‐isolated as Fusarium oxysporum Schlecht. f. sp. melongenae (Fomg) as evidenced. These isolates from weed hosts were assigned to VCG 0320. This study is the first report of Fomg isolated from weeds in eggplant fields in Turkey. None of the weed species tested showed symptoms of wilting in pot experiments, and F. oxysporum was isolated with greater frequency from all inoculated weeds. The results of this study indicate that several weed plants may serve as alternative sources of inoculum for Fomg, during the growing season.     

Decomposition of Plant Debris by the Nematophagous Fungus ARF

In the study of the biological control of plant-parasitic nematodes, knowledge of the saprophytic ability of a nematophagous fungus is necessary to understand its establishment and survival in the soil. The objectives of this study were (i) to determine if the nematophagous fungus ARF (Arkansas Fungus) shows differential use of plant residues; and (ii) to determine if ARF still existed in the soil of a field in which ARF was found originally and in which the population level of Heterodera glycines had remained very low, despite 15 years of continuous, susceptible soybean. Laboratory studies of the decomposition of wheat straw or soybean root by ARF were conducted in two separate experiments, using a CO₂ collection apparatus, where CO₂-free air was passed through sterilized cotton to remove the microorganisms in the air and then was passed over the samples, and evolved CO₂ was trapped by KOH. Milligrams of C as CO₂ was used to calculate the percentage decomposition of the plant debris by ARF. Data indicated ARF decomposed 11.7% of total organic carbon of the wheat straw and 20.1% of the soybean roots in 6 weeks. In the field soil study, 21 soil samples were taken randomly from the field. Only 3 months after the infestation of the soil with H. glycines, the percentage of parasitized eggs of H. glycines reached 64 ± 19%, and ARF was isolated from most parasitized eggs of H. glycines. Research results indicated ARF could use plant residues to survive.     

Molecular Identification and Genetic Diversity of Fusarium species Associated with Onion Fields in Turkey

To identify Fusarium species associated with diseases of root and basal plate of onion, surveys were conducted in seven provinces of Turkey in 2007. Samplings were performed in 223 fields, and 332 isolates belonging to 7 Fusarium spp. were obtained. The isolates were identified as F. oxysporum, F. solani, F. acuminatum, F. equiseti, F. proliferatum, F. redolens, and F. culmorum based on morphological and cultural characteristics. Also, species‐specific primers were used to confirm the identity of Fusarium species. F. oxysporum was the most commonly isolated species, comprising 66.57% of the total Fusarium species. F. redolens was identified for the first time in onion‐growing areas of Turkey. Selected isolates of each species were evaluated for their aggressiveness on onion plant. F. oxysporum, F. solani, F. acuminatum, F. proliferatum, and F. redolens were highly pathogenic, causing severe damping‐off on onion plants cv. Texas Early Grano. Inter‐simple sequence repeats (ISSR) markers revealed a high degree of intra‐ and interspecific polymorphisms among Fusarium spp.     

Morphological and Molecular Characterization of Fusarium Isolated From Maize in Syria

Maize is the third most important cereal after wheat and barley in Syria. Maize plants are attacked by several Fusarium species causing mainly stalk and ear rot of maize which poses a major impact worldwide. Identification of Fusarium species is important for disease control and for assessment of exposure risk to mycotoxines. To identify Fusarium species attacking maize in Syria, a total of 32 Fusarium isolates were recovered from maize ears collected from four different geographical regions, mainly from Ghouta surrounding Damascus. Fusarium isolates were identified based on morphology and on partial DNA sequencing of the TEF1‐α and rDNA/ITS genes. The majority (26 of 32) of these isolates was identified as F. verticillioides (subdivided into four groups), whereas three isolates turned out to be F. thapsinum, F. equiseti and F. andiyazi. The remaining three isolates were close to F. andiyazi, although further investigation is needed to confirm whether they represent a yet undescribed species. Furthermore, our results showed that sequencing the TEF1‐α gene is much more informative than sequencing of the rDNA/ITS region for Fusarium identification at the species level. PCR analysis showed that only F. verticillioides isolates were potentially fumonisin producers and that only the F. equiseti isolate was potentially trichotecene producer. This is the first report on Fusarium thapsinum, F. equiseti and F. andiyazi attacking maize in Syria.     

Molecular Identification,Mycotoxin Production and Comparative Pathogenicity of Fusarium temperatum Isolated from Maize in China

A recently isolated Fusarium population from maize in Belgium was identified as a new species, Fusarium temperatum. From a survey of Fusarium species associated with maize ear rot in nineteen provinces in 2009 in China, ten strains isolated from Guizhou and Hubei provinces were identified as F. temperatum. Morphological and molecular phylogenetic analyses based on the DNA sequences of individual translation elongation factor 1‐alpha and β‐tubulin genes revealed that the recovered isolates produced macroconidia typical of four‐septate with a foot‐shaped basal cell and belonged to F. temperatum that is distinctly different from its most closely related species F. subglutinans and others within Gibberella fujikuroi complex species from maize. All the strains from this newly isolated species were able to infect maize and wheat in field, with higher pathogenicity on maize. Mycotoxin determination of maize grains infected by the strains under natural field condition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry analyses showed that among fifteen mycotoxins assayed, two mycotoxins fumonisin B₁ and B₂ ranging from 9.26 to 166.89 μg/g were detected, with massively more FB₂ mycotoxin (2.8‐ to 108.8‐fold) than FB₁. This mycotoxin production profile is different from that of the Belgian population in which only fumonisin B₁ was barely detected in one of eleven strains assayed. Comparative analyses of the F. temperatum and F. subglutinans strains showed that the highest fumonisin producers were present among the F. temperatum population, which were also the most pathogenic to maize. These results suggested a need for proper monitoring and controlling this species in the relevant maize‐growing regions.     

植物群落物种多样性研究综述

物种多样性是生物多样性在物种水平上的表现形式 ,包括两方面的含义 ,一是指一定区域内物种的总和 ,主要从分类学、系统学和生物地理学角度对一个区域内物种的状况进行研究 ,也称区域物种多样性 ;二是指生态学方面物种分布的均匀程度 ,常常是从群落组织水平上进行研究 ,也称为生态多样性或群落多样性[1] 。本文所涉及物种多样性即为群落组织水平上的物种多样性。植物群落物种多样性的研究是其它多样性 (遗传多样性、生态系统多样性等 )的基础 ,有大量的研究成果相继报道 ,也有一些综述对植物群落物种多样性某一领域的研究进行总结。Ｍａｇｕ…     

陆地植物群落物种多样性演替研究进展

植物群落物种多样性在不同梯度上,其变化规律不一致.物种多样性与纬度呈反相关关系,与水分关系密切,表现出不同变化规律,与海拔主要表现为反相关关系和中间高度膨胀规律.土壤盐度、水分、肥力对物种多样性也有影响.在不同演替阶段物种多样性出现不同变化规律,群落结构最复杂的演替阶段,物种多样性最高.     

Mycoflora of Iranian Maize Harvested in the Main Production Areas in 2000

Maize is one of the most important cereals produced in, and imported into, Iran. The incidences of seed-borne fungi were determined in Iranian maize harvested in 2000 from four major production areas with different climatic conditions, namely Fars, Khuzestan, Kermanshah and Mazandaran provinces. This is the first study to compare the mycoflora of maize in the aforementioned areas. Mycological analyses showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species (83% of Fusarium isolates and 52% of the total isolations), with the highest incidence in Mazandaran (59%), a region of Iran with the highest rainfall and relative humidity, high rate of esophageal cancer (EC) and high levels of fumonisins in maize. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus. The incidence of A. flavus was highest in Kermanshah, the province with lowest mean minimum temperature. Penicillium species were seen in all the samples and Fars had the highest incidence, with highly significant differences when compared to the other three provinces. Diplodia species were not isolated from any of the samples examined.