Identification And Biological Characteristics Of Two New Cyst-forming Nematodes Species In Alpine Meadow Of Tianzhu,Gansu Province

Cyst-forming nematode is a kind of plant parasitic nematodes with strong adaptability,wide host and distribution which can cause economic losses to important crops.Presently,the studies on cyst nematodes in alpine meadow are very scarce.Therefore,this study investigated the occurrence and distribution of cyst-forming nematode in alpine meadow of Gansu province,described two new species of cystforming nematode parasitic on grass,and studied their biological characteristics,parasitism of legume crops and potential distribution.The results are as follow:1.During 2018 to 2020,random sampling method was used to investigate the cyst nematode of grass in Tianzhu alpine meadow.The results showed that: 5 towns including 34 villages,236 samples(including 77 cyst-forming nematode samples)were collected,the detection rate of cyst nematodes was 32.63%,The rate in Zhuaxixiulong is highest at 57.7%,the average density of cysts in Yongfeng village was highest(per100 g sample containing cysts in soil was 30.25).Total 12 families and 21 kinds of forage grass were collected,we found the rhizosphere soil of M.sikkimensis and P.viviparum with a higher cysts base and the highest density of cysts reached per 100 g sample soil contains and 61 and 31 cysts,respectively.2.Heterodera microulae sp.n.was isolated from the roots and rhizosphere soil of Microula sikkimensis in China.Morphologically,the new species is characterized by lemon-shaped body with an extruded neck and obtuse vulval cone.The vulval cone of the new species appeared to be ambifenestrate without bullae and a weak underbridge.The second-stage juveniles have a longer body length with four lateral lines,strong stylets with rounded and flat stylet knobs,tail with a comparatively longer hyaline area,and a sharp terminus.This species belongs to the Goettingiana group in the genus Heterodera.Compared with 7 valid species in this group,the length of fenestration and vulval slit were smaller than these species,however,the J2 body length and the MB were larger.The phylogenetic analyses based on ITS-r DNA,D2-D3 of 28 S r DNA,and COI sequences revealed that the new species formed a separate clade from other Heterodera species in Goettingiana group,which further support the unique status of H.microulae sp.n.Therefore,it is described herein as a new species of genus Heterodera.3.Cactodera tianzhuensis n.sp.was isolated from the rhizosphere soil of Polygonum viviparum.Morphologically,the new species is characterized by lemonshaped or rounded cysts that have protruding necks and vulval cones.The vulval cone of the new species appeared to be circumfenestrate without bullae and underbridge,vulval denticle present and anus distinct.Second-stage juveniles are vermiform,stylet well-developed with the rounded stylet knobs to slightly concave anteriorly.Lateral field with four incisures.Tail gradually tapering to a finely rounded terminus,outline of hyaline portion is V-shaped or U-shaped.This species is most similar to C.thornei,but differs from eggshell surface(smooth vs punctate).The phylogenetic analyses based on ITS-r DNA,D2-D3 of 28S-r DNA clearly revealed that the new species formed a separate clade from other Cactodera species,which further support the unique status of C.tianzhuensis n.sp.Therefore,it is described herein as a new species of the genus Cactodera.4.The biological characteristics of H.microulae and C.tianzhuensis were studied respectively.The results showed that the optimum incubation temperature of two species were 15? and 20?,respectively.For two species,soil immersion and dilution of 20 times root juice can promote eggs hatching,but 5 times root juice inhibited.Second-stage juveniles of H.microulae had higher viability and activity at 15?,while C.tianzhuensis had higher viability at 18?.Second-stage juveniles of both species could be better preserved at 4?,but the activity of J2 s were weak.The life cycle of H.microulae is 27 d.5.The host suitability and potential region of H.microulae were analyzed.The results as follow: J2 s can invade pea,cowpea,kidney bean,mung bean and faba bean,the number of J2 s was the most in the roots of faba bean and least in cowpea.Browning reaction was found using acid staining strong.After 60 days,cyst nematodes were detected in faba bean.The morphometrics of faba bean population were lower than original population unless cyst size.Max Ent analysis showed that the potential distribution of H.microulae are central region in Gansu,eastern region in Qinghai,central region in Tibet.When the wettest monthly precipitation is about 98 mm,the highest probability of H.microulae exists,the other important environmental factors as follows: the wet season is about 240 mm rainfall,the most warm precipitation is about200 mm,as well as the wet season average temperature of 11.5 ?.