Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T₁, T₂ and T₃ transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

     

Feasibility of growing Moringa oleifera as a mix-crop along with tomato for control of Meloidogyne incognita and Rotylenchulus reniformis in Egypt

Impact of nematicidal potential of drumstick, Moringa oleifera Lam. (Moringaceae), as a medicinal plant (1–4 plants per pot) is obtained when it is used as a mix-crop along with tomato cv. Super Strain B against Meloidogyne incognita and Rotylenchulus reniformis was evaluated under a greenhouse conditions (30?±?5?°C) at the National Research Centre, Egypt. The nematodes final population and their rate of build-up as well as the root gall index were significantly affected by the number of moringa plants when grown with tomato together. There was a negative correlation between the number of moringa seedlings and the final population of both nematodes. The lowest nematode final population and rate of build-up were determined at the highest number of moringa plants (four plants per pot). The highest number of root gall index (4.1) was found on roots of tomato grown alone, while, the lowest one (0.6) was found on roots of tomato grown with four plants of moringa. This type of control is considered easy, inexpensive and pollution-free.     

Comprehensive report on the prevalence of root-knot nematodes in the Poonch division of Azad Jammu and Kashmir,Pakistan

The present study documents the root-knot nematodes (RKN) fauna of the Poonch division in Azad Jammu and Kashmir infecting vegetables. An overall prevalence of 40% of RKN was recorded. Of the four districts investigated, maximum prevalence was recorded in district Poonch with 59%, followed by Sudhnuti with 58%. The lowest prevalence of RKN was found in districts Bagh (29%) and Haveli (33%). Out of 15 vegetables investigated, RKN was found on five crops. The highest prevalence of 37.8% was recorded on okra, followed by 31.3% on cucumber and 17.5% on tomato. RKN was less prevalent on eggplant (8.3%) and beans (7.7%). Three RKN species, that is Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria, were found infecting the hosts. M. javanica was found to be the most prevalent followed by M. incognita and M. arenaria. This trend was found in all the districts. Overall prevalence of M. javanica as sole population was 9% and that of M. incognita was 2%. Meloidogyne arenaria was not found in any of the fields as sole population. The prevalence of M. incognita with M. javanica or M. arenaria as mixed populations was 8% and 5%, respectively, and that of M. javanica with M. arenaria was 4%. Similarly, all the three species prevailed as mixed populations in 12% of the fields in the division. The severity of RKN infections, measured as galling index, was found to be variable within each infected field (GI 2–9). Identification of RKN species was based on the morphology of perineal patterns and confirmed by molecular SCAR and CO1 makers based identification. In conclusion, RKN were distributed in the Poonch division and M. javanica was predominant. Cucumber, okra, tomato and eggplant were severely attacked by these nematodes warranting the adoption of stringent control strategies for their management.     

Involvement of salicylic acid in induction of nematode resistance in plants

The role of salicylic acid (SA) as a possible signaling component in the case of the infection of plants with nematodes has been studied using a model system consisting of the tomato (Lycopersicon esculentum (Mill.) and race 1 of the gall eelworm Meloidogyne incognita (Kofoid and White, 1919; Chitwood, 1949). The preplanting SA treatment of tomato seeds results in an increased nematode resistance of susceptible tomato cultivars; the protective effect is higher in the case of SA combined with chitosan, a biogenic elicitor of plant resistance. The studied preparations stimulate the growth and development of the plants. The increase in the resistance of tomato plants is related to the increased activity of phenylalanine ammonia-lyase and an increased SA content in plant tissues infected with nematodes; both these factors significantly influence nematode development.     

<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Management of Fusarium oxysporum f. sp. lycopersici and root-knot nematode disease complex in tomato by use of antagonistic fungi,plant resistance and neem

Simultaneous infestation with root-knot nematodes (RKN) and Fusarium oxysporum f. sp. lycopersici (FOL) leads to formation of a disease complex that increases crop losses than effect of either RKN or FOL. In this study a management programme involving plant resistance, biological control agents, and neem was carried out to manage RKN and fusarium wilt disease complex. The biological control agents were Purpureocillium lilacinum (PL) and Trichoderma harzianum (TH) while the RKN was Meloidogyne javanica. In vitro dual culture plates were set up to test the interaction of biological control agents and FOL. Greenhouse experiments were conducted using two tomato cultivars Rambo F1 and Prostar F1. The treatments were; PL, TH, PL–TH, neem, PL neem, TH neem, and PL–TH neem. Each treatment was replicated four times and the treatments set up in a randomised complete block design in the greenhouse. Inhibition of FOL mycelial growth by TH and PL was 51.9%, and 44% respectively by the ninth day in vitro culture plates. In the cultivar, Prostar F1, the treatments PL–TH, PL, and TH in the presence or absence of neem had a FOL disease severity score significantly lower than the untreated control. Host resistance sufficed to prevent infection of Rambo F1 with FOL. The treatments PL–TH, PL and TH reduced FOL propagules and M. javanica juveniles in the roots and performed even better when combined with neem in both tomato cultivars. Therefore, a host that is resistant combined with biological control agents and organic amendments can be used in the management of RKN and FOL in tomato production.     

ToMV-Resistant transgenic tomato as a material for the first field experiment of genetically engineered plants in Japan

Summary An F₁ hybrid betweenLycopersicon esculentum andL. peruvianum was transformed using a Ti-plasmid binary vector with a coat protein gene cDNA of an attenuated tomato mosaic virus (ToMV) strain L₁₁A which was expressible by the 35S promoter of cauliflower mosaic virus (CaMV). A transgenic plant which expressed the most resistance to ToMV was chosen as a material to be tested in a nonisolated greenhouse and in the field. This transgenic tomato plant was propagated by cutting. In the first test using an isolated greenhouse and in the second test conducted in an nonisolated greenhouse, no major morphologic and physiologic differences were found between the transgenic plants and the nontransgenic control plants. Also, there was no evidence that the transgenic plants produced any new hazardous substances. Both the transgenic and the nontransgenic plants were self-sterile, and crossing of the cultivated species with pollen of these plants produced few seeds. These features of the transgenic plants satisfied the requirements for a small scale field test. The field test of the transgenic plants are in progress. Presented in the Session-in-Depth “Field Test Requirements and Performance of Transgenic Plants” at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

丛枝菌根真菌对不同番茄品种抗根结线虫病的影响

【背景】根结线虫病害严重制约我国设施蔬菜的生产。丛枝菌根真菌(Arbuscular Mycorrhizal Fungi,AMF)作为土壤中最重要的有益真菌之一,可以促进植物生长,提高植物抗病性,减轻土传真菌和线虫病害的发生。在蔬菜保护地栽培中,AMF对于植物线虫病防治作用的研究受到了广泛关注。【目的】针对番茄生产中危害最严重的南方根结线虫(Meliodogyne incognita)病害问题,研究AMF和番茄品种不同组合的抗线虫效应,以期为菌根真菌作为生物防治剂和生物菌肥应用于实际生产提供技术基础。【方法】在灭菌土壤中,人工接种根结线虫,比较不同菌种Rhizophagus intraradices(Ri)、Acaulosporamellea(Am)及菌种组合Rhizophagusintraradices+Acaulosporamellea(Ri+Am)在不同番茄品种(感病品种蒙特卡罗和抗线虫品种仙客1号)上对南方根结线虫侵染和繁殖的影响,研究AMF对根结线虫的拮抗效应。另外,采用南方根结线虫连作发病的土壤,在感病品种蒙特卡洛上接种AMF混合菌种Ri+Am,番茄苗移栽入连作土壤中,测定各生长指标和调查根结和卵块数量,评价接种AMF处理对根结线虫病的防治效果。【结果】在灭菌土壤中,普通番茄品种蒙特卡罗的菌根效应显著优于抗线虫番茄品种仙客1号,表现为前者单位根重的根结和卵块的数量均比对照显著降低,而后者仅降低了卵块数量;蒙特卡罗上接种Ri+Am混合菌种的效果优于接种单一菌种Am和Ri;而仙客1号上接种Ri的效果更好。接种线虫也显著影响了AMF的侵染,而且对抗性品种仙客1号的影响更为明显。但除了接种Am的处理,大多数处理收获时菌根侵染率仍维持较高的水平(70%以上)。在连作土壤中,感病品种蒙特卡罗接种混合菌种Ri+Am具有较好的抗/耐线虫效应,主要表现为促进植株生长,显著降低根结和卵块数量,但菌根侵染率较灭菌土壤低,约为40%。【结论】综合以上结果,表明菌根化苗能够在一定程度上减轻根结线虫病的危害。土壤灭菌条件下,在感病和抗线虫番茄品种上接种AMF能够减轻线虫的侵染和繁殖,而且在感病品种上的防治效果更加显著。在连作土壤中,在番茄感病品种上接种AMF也表现较好的抗线虫效果。     

Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Candidate genes underlying the quantitative trait loci for root-knot nematode resistance in a Cucumis hystrix introgression line of cucumber based on population sequencing

The southern root-knot nematode (RKN), Meloidogyne incognita (Kofoid & White) Chitwood, is one of most destructive species of plant parasitic nematodes, causing significant economic losses to numerous crops including cucumber (Cucumis sativus L. 2n = 14). No commercial cultivar is currently available with resistance to RKN, severely hindering the genetic improvement of RKN resistance in cucumber. An introgression line, IL10-1, derived from the interspecific hybridization between the wild species Cucumis hystrix Chakr. (2n = 24, HH) and cucumber, was identified with resistance to RKN. In this study, an ultrahigh-density genetic linkage bin-map, composed of high-quality single-nucleotide polymorphisms (SNPs), was constructed based on low-coverage sequences of the F_2:6 recombinant inbred lines derived from the cross between inbred line IL10-1 and cultivar ‘Beijingjietou’ CC3 (hereinafter referred to as CC3). Three QTLs were identified accounting for 13.36% (qRKN1-1), 9.07% and 9.58% (qRKN5-1 and qRKN5-2) of the resistance variation, respectively. Finally, four genes with nonsynonymous SNPs from chromosome 5 were speculated to be the candidate RKN-resistant related genes, with annotation involved in disease resistance. Though several gaps still exist on the bin-map, our results could potentially be used in breeding programs and establish an understanding of the associated mechanisms underlying RKN resistance in cucumber.

     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.     

Spectrum of the Ma genes for resistance to Meloidogyne spp. in Myrobalan plum

The Myrobalan plum, Prunus cerasifera, bears a complete-spectrum resistance to the root-knot nematodes (RKN) Meloidogyne spp. in comparison to the main resistance sources in Amygdalus rootstocks that have more restricted spectra, as evidenced by a differential resistance test based on the predominant species M. arenaria, M. incognita and M. javanica and the population M. sp. Floride. Resistance to M. arenaria (A) in Myrobalan plum is controlled by the Ma major resistance genes that are completely dominant and confer a non-host behaviour that totally prevents the multiplication of the nematode. The inheritance of resistance of this self-incompatible species to M. incognita (I), M. javanica (J) and the population M. sp. Floride (F), considered as belonging to a new RKN species, was studied using G₁ hybrids from a diallel cross based on five parents, the two resistant P.2175 (Ma1 gene; heterozygous) and P.1079 (Ma2 gene; homozygous) and three host parents, P.2032, P.2646 and P.16.5 (recessive for both genes), completed with the G₂ backcrosses P.16.5×(P.2646×P.1079), P.2646 ×(P.16.5×P.1079) and P.2175×(P.2646×P.1079). G₁ and G₂ clones obtained from softwood cuttings sampled from trees in the field experimental design, rooted in the nursery, and inoculated in containers (six replicates per clone) under greenhouse conditions, were simultaneously evaluated for their host suitability to two to four of the RKN species, based on a 0–5 gall index (GI) rating under a high and durable inoculum pressure of the nematode, and then classified into resistant (R; GI?0.2) or host (H; GI?1.3) classes. The resistance classification of each individual clone, evaluated to two (A/J: 319 clones), three (A/J/I: 249 clones) and four (A/J/I/F: 161 clones) RKN species, from segregating and non-segregating crosses involving either Ma1 or Ma2 or both or none, was identical whatever the species. The independence of the R/H classification from the tested RKN indicates that the Ma1 and Ma2 genes control resistance to all of them, and it is assumed that these genes also control resistance to other minor RKN species. The relationship of the Ma genes with the putative genes involved in Amygdalus sources is discussed with the objective of introducing them into new interspecific rootstocks expressing a complete-spectrum and high-level resistance.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Pathogenicity of Pochonia species on eggs of Meloidogyne javanica

Among fungi, species of the genus Pochonia Batista & O.M. Fonseca are considered as promising biological control agents with high potential to reduce root-knot nematode (RKN) and nematode populations. In this research we investigated Fars province of Iran for the presence of Pochonia spp., compared pathogenicity of different Pochonia species on eggs of RKN in vitro, and selected the best isolates for further studies. During 2004-2006, 128 soil samples of fields infested with cyst nematodes and 18 soil samples infested with RKN were collected from Fars province of Iran. In vitro pathogenicity tests were carried out on 36 isolates of Pochonia spp. obtained from CBS and IRAN culture collections. The seven best isolates of this experiment were selected for greenhouse test and their ability in controlling RKN was examined in natural soil. In greenhouse test fresh weight of plant’s tops and roots, gall index, nematode multiplication, second-stage juveniles’ population in soil, reproduction rate (P_f/P_i), proportion of infected eggs, control efficacy, root colonization and soil colony forming units were determined. In vitro pathogenicity of Pochonia on RKN eggs varied between 39% and 95% eggs infected. In greenhouse experiment, three isolates are promising for control of RKN and selected isolates are subjected to more extensive testing to determine their effectiveness in a range of conditions before being developed as commercial biological control agents.     

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H₂O₂ and O₂ ^−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P _n) and the maximal photochemical efficiency of PSII (F _v/F _m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.     

Antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (<Emphasis Type="Italic">LeFAD7</Emphasis>) enhances the tomato high-temperature tolerance through reductions of trienoic fatty acids and alterations of physiological parameters

We studied how the reductions of trienoic fatty acids (TAs) and increases of dienoic fatty acids (DAs) enhanced high-temperature tolerance in antisense expression of tomato chloroplast omega-3 fatty acid desaturase gene (LeFAD7) transgenic tomato (Lycopersicon esculentum Mill.) plants. In transgenic plants, the content of linolenic acid (18:3) was markedly decreased, while linoleic acid (18:2) was increased correspondingly and the similar changes were observed under high-temperature stress as well. Under high-temperature stress, transgenic plants can maintain a relatively higher level of net photosynthetic rate (P _N) and chlorophyll (Chl) content than that of wild type (WT) plants. A decreased Chl/Carotenoids (xanthophylls and carotenes, Car) ratio and Chl a/b ratio were observed in transgenic plants. Transgenic plants exhibited visible decrease in the relative electrolyte conductivity, higher activities of antioxidative enzymes and lower reactive oxygen species correspondingly than WT. In addition, high-temperature stress for 24 h caused more extensive changes of chloroplast ultrastructure in WT than in transgenic plants. We therefore suggested that the enhancement of high-temperature tolerance in antisense expression of LeFAD7 transgenic plants might be raised from the reduction of TAs and increase of DAs subsequently leading to series of physiological alterations.