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Abstract ~e present study was conducted during two consecutive seasons, 1985 and 1986 at the Faculty of Agriculture Moshtohor, Zagazig University, Kalubia Governorate. Bio_fertilization studies have called the attention toward soil microorganisms as a good alternative to chemical fertilization because of its cheep costs and it causes no pollution. Mycorrhizal fungi is considered as one of the biefertilizers which live between plant rootS. Consequently, this investigation was carried out, to study the effect of endomycorrhizal fungi inoculation and phosphorus fertilization on soil properties, infection and intensity, mycorrhizal dependency ratio (MDR) of rootstock, dry weight of different parts of seedlings, top/root ratio, root growth and distribution, leaf and root minerals content, leaf chlorophyll and carotene contents, leaf sugars and stem total carbohydrates, leaf and root amino acids content, and leaf cyto~inins content of two citrus rootstocks. Two-year-old seedlings of two citrus rootstocks, i.e. Cleopatra mandarin and sour orange were transplanted in woody boxes filled with clay loam soil disinfected with 2% Formalin solution (2 Seedlings per each box). The treatments used in this study involved I 1. Boxes left untreated as control. 2. Boxes fertilized with phosphorus at the rate of 5g P205 as superphosphate (control). 3. Boxes unfertilized with P but the soil Was inoculated with glEm~ m~££E£§rp~fungi. 4. Boxes unfertilized with P but soil was inoculated with Glo.!!\l~1..!§!!i~£~ fungi. 5. Boxes fertilized with phosphorus at the rate of 5g P205 as superphosphate and the soil Was inoculated with g12m~.§m~££9~§EPus fungi. 6. Boxes fertilized with phosphorus at the rate of 5g P205 as superphosphate and the soil Was inoculated with Q1Em~§ ~ustrale fungi. On the other hand, other set of seedlings were planted in 30 cm diameter clay pots treated with mycorrhizae and fertilized with phosphorus with the same rate for boxes for the infection and intensi’t7 stUdies. The obtained results could be summerized as follows : 5.1 IDfecUoD I 1. Mycelium and arbuscules of mycorrhizae fuugi on roots of Cleopatra mandarin and sour orange started with low percentages in May followed by a gradual increase in July to reach the maximum (l~) in September. However, in both rootstocks vesicles percentages on citrus roots were almost l~ in all sampling dates. 2. Mycelium, vesicles, and arbuscules on roots of control rootstocks used were nil. :3. In May and July, seedl~. inoculated with mycorrhizae fuugi and unfertilized with phosphorU8 gave hip;herpercentages of myc.eliWll aDd arbuscules on their roots as compared to that of inoclllated and fertilized ones. 4. Generally, in all sapling dates, $ll0!!l1lS austrw fwl8i was associated with higher percentages of arbusclllesthaD ’su’CPM 88&rocarplMl mycorrhi •••• 179 5.2. Intensity: 1. Number of mycelium. vesicles. and arbuscules on roots of the two studied citrus rootstocks started with low numbers in May followed by an increase in July and a sudden increase in September. 2. Citrus seedlings inoculated with mycorrhizae and unfertilized with phosphorus had roots with higher number of mycelium. vesicles. and arbuscules as compared with those treated with mycorrhizae and fertilized with phosnhorus. 3. Glomus australe fungi caused a hip;her increase in number of vesicles and arbuscules on rootl or o1trua rootltocks aore thaD ~QlY’ macrocarpus mycorrhizae. On the other hand, the effect of mycorrhizae species on ayc.li_ number depended upon the rootstock Wled. In this respect, whUe GlomUS aLl!trW funsi were superior in increasing number of mycelium on roots of Cleopatra mandarin seedl1Dgs as coapared to GlomW! macrocarpy!!. the picture was changed to the opposite when lour oraus. s.84- 1illgs were concerned. 5.3. ~corrhizal dependency ratio (1IlJil) I 1. Cleopatra mandarin dependecl51.ightly_ on mycorrhizae than sour orange rootstock. 2. QJ,0l!I!.l1 .9lacfocarplWf’UJ:l6iwas more effective on MDR of seedlings than the .Ql..£I!l.9! austral,!! fungi. 3. Applying phosphorus to citrus seedlings decreased visually MDR as compared to unfertilized plants. 1. Higher Values of dry weight parameters were observed for sour orange seedlings as compared to Cleopatra mandarin. 2. Mycorrhizae fungi treatments increased cU7 weight of different parts of seed.l1np:s and top/ root ratio as compared to control plants • .ilO!!lW! lIacfocarpW! flUUl:iwas 1I0re prOlllis1ng in iDCreasi~ dr;y weights of whole seed.lilUl., leaves, stem and roots than ’suQ!I,Y! ,,,,trw. Such reSQIt was not clearly noticed ror top/ root ratio. 3. Applying phosphorus only to citrus Seed- 1111gscaused. s1gn1finant 1Dcreue ill cU7 . weight of roots whereas other parameters of dry weight used in this study were decreased under phosphorus fertilization. 5.5. Boot growth : 1. Cleopatra mandarin and sour orange seedlings unfertilized with phosphorus and inoculated with lilolll,.mWa!crocarpus fungi gave higher Values of root growth expressed as root coefficient. 2. Cleopatra mandarin seedlings gave general.Q’ higher Values of root growth as cOlllparedto the analogouS ones of sour orange rootstock. 3. Generally as a Spec1tic effect of mycorrhizal fungi. Glomus austral, enhanced better root growth of citrus seedl~8 than GIO!i! iterooarpYi fun~i under the experimental 8011 environment. 4. Unfertilized seedlings with phosphorus 8urpas8ed in their root growth the P fertUize4 ones. 5.6. Root distribution , 1. Cleopatra mandarin seedlings inoculated With GIOJllusaustraJ& fungi and unt’ertilized with phosphorus was preferable cOJllbination for encouraging root distribution of plants among the other treatments used whUe sour orange seedlings received phosphorus and treated with GIOJllusmacrocarRB!! fungi surpassed all other treatments relDaTkably in this respect. 2. Cleopatra mandarin plants had higher number of 3-5 m.m. and 2-3 m.m. roots whereas sour orange seedlings were Superior in 1-2 m.m. roots and total number of roots. 3. Mycorrhizae fungi treatments increased s1g_ n1ficantly root number of different diameters and total root number. In the same time, ~!,y macrocarpy’! fungi wart more promising in increasing number of’1-2 m.m. and 3-5 a.m. roots. On the other hand, ~.Y§ ~.&U III¥corrhi••e f’ungi. encouraged developing of’2-3 m.m. roots and total number of’roots. 4. P unfertilized seedliDgs not only increased root number of different diameters but also the total number of roots than fertilized seedlings. 5.? Lea:t1Il1neJ.’aclosntent : 1. Generally 10weJ.’amounts of N and Ca and were higher levels of K and Zn/existed in leaves of sour orange rootstock as compared with those of Cleopatra mandarin. No statistioidifference between the two rootstocks used was observed concerniDg leaf P and Mg contents. 2. Mycorrhizae species raised up Leaf N. P, I, and Kg contents and decreased lea:t Oa and 2Q contents in respect of contJ.’olplants. GlaRy! austrai§ fungi were more positive in stimulating leaf p. K. and Mg contents than Gl!Ml\Llm!acJ.’ocarpus spec~e. The significant difference was lackiDg in case of leaf N and Oa contents. 3. Phosphorus fertilizer eitheJ.’added for SOUl.’ oJ.’aDgeOJ.’Cleopatra mandarin seedlings increased lea:t p. K. and Oa contents and decJ.’easedlea:t _, JIg. and Zn l.”els. 5.8. Root minerals content I 1. Control seedliDKs fertilized with phosphorus had roots with higher amounts of N and P and lower levels of K and Mgas coapared with unfertilized control plants. No clear trend or difference was noticed in case of zn anrl Ca nutrients. 2. Roots of sour or~e seedliDKs contained higher amounts of N, K, Ca, Mg, and zn than those of Cleopatra mandarin seedliDKs which were more rich in root P content. 3. ~corrhizae fungi decreased root P, Ca, Mg, and zn contents and in the S8llletime increased JIT and K levels as compared with non inoculated seedlings (control). §lomus austrw flUlgi had increased root N and zn contents aDd decreased root P, K, Ca, and JIg levels. lio significant difference was observed betweeo the two species of I117corrhiz.e in their effect on root minerals content. 4. Phosphorus fertilization 1Dcreased JIT aDdP contents of citrus rootstocks whilst it had no effect on root K, Ca, Mg, and 2’.D since the difference was small. 5.9. Leaf chlorop~ll and carotene contents : 1. Leaves of sour orange seed1iDP;scontained higher amounts of chlorop~ll (A) and total chlorophyll and lower level of carotene in respect of Cleopatra mandarin. Citrus sPecie. s had no statistical effect on leaf chlorophyll (B). 2. Mycorrhizae species used were promising in building up more leaf chlorophyll and carotene over the control. Meanwhile. G10ll1!laSustaale 1’uIlll:ias comparedwith Glom!lSmacrocarpys decreased leaf chlorophyll (A) and carotene whilst they increased leaf chlorop~ll (B) and total chloropb.vll. Such effect was not statistical17 observed in case of leaf total chlorop~ll. 3. ceedlings receiving phosphoru. were iD1’erior in their leaf chlorop~ll and carotene contents. However, significant di1’1’ereaces were lacking whenleaf chlorop~ll (B) end carotene contents were concerned. 186 5.10. Leaf susars and stem total carbohydrates : 1. Sour orange seedlings had leaves with higher amounts of non redu~and total susars as iliellas stem total carbohydrates in respect of those of Cleopatra mandarin. Leaf reducUg sugars of sour orange wereslightly less than those of Cleopatra mandarin. 2. ,@,omlas.!,!ustralf,u§ngi decreased significantly leaf non-redu~ and total sugars and slightly reducUlg susars than the control. 3. Applying phosphorus to citrus plants induced a significant decrease in leaf non-reduciag sugars but it had no statistical effect on leaf reduciJtgand total sugars as well as stem total carbohydrat es• 5.11.Leaf nitrogen fractions : 1. Leaves of Cleopatra mandarin seedlillgs contained higher amounts of soluble nitrogen, rest nitrogen, and ammonium nitrogen and lower level of crystalloid nitrogen as compared with those of sour orange. Citrus rootstocks had no statistical effect on leaf nitrate content. 2. Comparing £llomus!!§.crocarpusfungi treatment with the control, it significantly decreased leaf crystalloid nitrogen and nitrate contents as well as increased leaf rest nitrogen but without any effect on leaf soluble nitrogen and ammonium nitrogen contents. Glomus aus~.! fungi, from other hand, increased leaf crystalloid nitrogen and ammonium nitro~en and decreased leaf rest nitrogen and nitrate contents. 3. Phosphorus application decreased leaf soluble nitrogen, rest nitrogen and nitrate contents and increased leaf ammonium nitrogen. Phosphorus, by all means, had no effect on leaf CJ:,Ystall01d nitrop;en. 5.12 Boot nitrogen fract10DB : 1. Roots of Sour orange seedlinp;swere more rich in soluble nitro~en and poor in nitrate content in respect of Cleopatra mandarin. No visual difference was observed between sour orange and Cleopatra mandarin regarding root crystalloid nitrogen, rest nitr08en, BDd emaonium nitrogen confents. 2. )fycorrhiaae 1’uDgifluctuated in their e1’fec_ on root nitrogen fractions content. In this concern, Glomus australe fungi succeeded in increasing root soluble nitrogen, cr,ystal10id nitrogen, rest nitrogen, and nitrate contents whereas ~s macrocarRU’ fungi raised up only root rest nitrogen, and nitrate contents. Both two mycorrhizae fungi used in this study failed to increase root ammonium nitrogen over the control. 3. Adding phosnhorus to citrus seedlinp;s increased only root nitrate content whilst it decreased the other root nitrogen tractions determined in this study. 5.13 Leaf and root •.ino acids conten’ I 1. Cleopatra mandarin seedlings generally gave hill:hervalues of leaf and root _inc acids content than those of sour orange. 2. l’ unfertilized but mycorrhisae inoculated plants of both rootstocks were hisher in their values of leaf •.ino acids content than corresponding uni.noculated ones.1he opposite w•• t~Qe whan ~corrhi..u fllDgi and fertilized seedlings were caapared with fertilized control ones. 3. Glomusaustral,!! fu.ogi resulted in an increase in leaf amino acids content than Glomusmacroc~ mycorrhizae. 4. Leucine and Isoleucine, Proline, and Hydro.xyProline existed in citrus leaves with higher amounts whereas leaf Valine, T,yrosine, Threonine, Glysine, Arginine, and Histidine showedan opposite trend. 5. Cleopatra mandarin and sour orange seedli. ogs fertilized with phoRohorus and inoculated with Glom~ macrocarpul fungi gave higher values of root amino acids content than those unfertilized and inoculated with the seme mycorrhizae fungi. 6. M3’corrhizae fungi treatments increased root amino acids level as COJllparedto non-inoculated control plants. At all ”’ents, GJ.oags aac£OCarpg fungi were more promisi.og in increasiug root emino acids content than GleaM austrMe. 7. !pplyiDg phosphorus to citrus seedlings induced a general inerease in root -.ino acids content as coapared with unfertiliZed and. !lbat was true in either inoculated or non-inoculated plants. 8. Leucine and Isoleucine. Proline. and Uydroxy Proline were hi~her in roots of citrus seedli~s wnilst Valine. ~hreonine. Glysine. Arginine. and Histidine were inferior in roots of citrus rootstocks studied in this research. 5.14 Leaf cytokiniDa content I 1. .Applying phosphorus to control seedliJ:Igs decreased leaf cytokinins content in June but in September the effect was changed to the reverse. Such result was more clear in case of Cleopatra mandarin rootstock. 2. Inoculati~ P unfertilized plants With mycDrrhizae increased leaf cytokinins content in both June and September samples. In sour orange rootstock such effect was not so 8tro~ as in Cleopatra mAndarin. ,. jpplyinl ph08phoru8 to inoclllatecl8Hdlqa of both citru. rootstocks iDCre ••eclleat c71iokinins content in Sept_ber as coapered with P unfertilized and inoculated plants. The picture was chaDKedto the reverse in June. 4. GlomuslIlacrocarpus fungi raised up leaf total cytokinins content in June in respect of GlolllQsaustrale fungi. The opposite was generally true whenleaves were sampled in Septelilber. 5. Leaf total cytokinins content was highest in June then decreased r6lllarkably in Sept6lllber. 5.15 8011 propertle. I 1. At the termination of the experilllent, analysis of soil showedthat phosphorWifertUization for Cleopatra lIlandarin resulted in an increase in available N and a.decrease in F, K, Ca, and 1IIg nutrients. In the case of sour orange, pho- 8~horus fertilization decreased 80il available N, P, and Ca contents and. increased avaUable K. 2. Adding G1Cll!RlIulascrocarpWlfuugi for unfertilized soil of Cleopatra lIlandarin 1J:Iducedan increase in its contents of avaUable 1f, P, and Ca and a decrease in soU K 8Dd Irs levels. In addition. Glomus aqstrale was superior in increasing soil available nutrients discarding P. Moreover. inoculating unfertilized soil of sour orange with mycorrhizae generally increased available macro-nutrients in the soil. 3· Soil fertilized with phospho~us and treated with Q!omu§ mac~carpus fungi contained higher levels of available N. p. and Ca. Similarly. m.~ aqstral! mycorrhizae caused an increase in soil available N. p. and Ca contents and a decrease in soil K level. mandarin and Sour orange Generally. inoculation of Cleopatra/ seedlings grown in stere1ized soil with endomycorrhizal fungi enhanced vegetative growth. root growth and distribution. and Chemical constituents of bo~h Cleopatra mandarin and sour orange seedl1nP:s. lPnrthemore • .9.lC!llH! australe fungi and no phosnhorus fertilization for Cleopatra mandarin seedlings and Gl.~ ,acrocarpus fungi and no phosnhorus fertilization for sour orange plants gave the best results of seedlings growth. Therefore. Gl.qs maerocarPUI or .§l-BI llWIYW fun~i could be used as bio-fertilization for citrus seedlings in fuaigated clay loam soil for producing good seedlings free from diseases. |